Compositions and Methods for Identifying Secretory Antibody-Bound Microbes

ABSTRACT

The invention relates to the identification of secretory antibody-bound bacteria in the microbiota in a subject that influence the development and progression of inflammatory diseases and disorders. Thus, the invention relates to compositions and methods for detecting and identifying the constituents of a subject&#39;s microbiota, methods of modifying the constituents of the microbiota, and methods for treating inflammatory diseases and disorders in a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is entitled to priority to U.S. ProvisionalApplication No. 61/777,519, filed Mar. 12, 2013, which is incorporatedby reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number2T32AR007107-37 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

The composition of the intestinal microbiota varies substantiallybetween individuals and has dramatic effects on host physiology anddisease susceptibility (Lozupone et al., 2012, Nature 489:220). A majormechanism by which the microbiota impacts the host is through itsinteractions with the intestinal immune system. Select members of themicrobiota exert dramatic effects on the intestinal immune system anddisease susceptibility through chronic stimulation of specific immuneresponses (Blumberg and Powrie, 2012, Science translational medicine4:137rv7; Chow et al., 2011, Current opinion in immunology 23:473;Hooper et al., 2012, Science 336:1268; Littman and Pamer, Cell host &microbe 10:311), which can be both beneficial and detrimental. In mice,for example, Clostridia species induce the expansion of regulatory Tcells and suppress allergic responses and intestinal inflammation(Atarashi et al., 2011, Science 331:337), Segmented Filamentous Bacteria(SFB) induce T helper 17 responses, exacerbate the development ofarthritis and protect against the development of diabetes (Wu et al.,2010, Immunity 32:815; Ivanov et al., 2009, Cell 139:485; Kriegel etal., 2011, Proceedings of the National Academy of Sciences of the UnitedStates of America 108:11548), and Bacteroides fragilis induces IL-10production by T helper cells and ameliorates intestinal inflammation(Mazmanian et al., 2008, Nature 453:620).

Alterations in the composition of the microbiota, sometimes referred toas “dysbiosis,” are known to drive development of both inflammatory andnon-inflammatory diseases including inflammatory bowel disease,metabolic diseases, and autoimmunity (Littman and Pamer, 2011, Cell Host& Microbe 10:311-323). Crohn's disease is one such example, and it ischaracterized by chronic inflammation of the intestinal tract andaffects millions of people worldwide (Abraham and Cho, 2009, New Engl.J. Med. 361:2066-2078). Although the exact cause of Crohn's disease isnot known, members of the intestinal microbiota are believed to play apivotal role in disease development. It is believed that many of thesediseases and disorders are driven by specific members of the microbiota,which are referred to as “pathobionts.”

Pathobionts are defined as bacteria present in the “normal” microbiotathat have the potential to cause or drive disease development, andtherefore share features with both commensal symbionts and pathogens(Chow et al., 2011, Curr. Opin. Immunol. 23:473-480). For example,Segmented Filamentous Bacteria (SFB) are common members of the mousemicrobiota that exacerbate the development of autoimmunity (Wu et al.,2010, Immunity 32:815-827), and Helicobacter species drive thedevelopment of colitis in genetically susceptible mice. SFB andHelicobacter species therefore represent classical pathobionts.

Immunoglobulin A is the predominant antibody isotype secreted into theintestinal lumen where it binds indigenous members of the microbiota andcontrols microbiota composition (Macpherson 2012, Immunological reviews245:132; Pabst, 2012, Nature Reviews Immunology; Suzuki et al., 2004,101:1981; Peterson et al., 2007, Cell host & microbe 2:328). While allintestinal bacteria can induce specific IgA responses in principle(Hapfelmeier et al., 2010, Science 328:1705; Macpherson et al., 2000,Science 288:2222; Macpherson and Uhr, 2004, Science 303:1662), directanalyses of the proportion of intestinal bacteria that are coated withIgA demonstrated that only a fraction of all intestinal bacteria aremeasurably IgA coated (Tsuruta et al., 2009, FEMS immunology and medicalmicrobiology 56:185; van der Waaij et al., 1996, Gut 38:348; van derWaaij et al., 1994, Cytometry 16:270). Because little is known about thespecificity of the intestinal IgA response in the context of a complexmicrobiota, whether this fraction is comprised of many species or a highpercentage of a few species remains unclear. However, while severalcommensal bacteria have been shown to induce specific IgA responses,pathobionts and pathogens induce higher levels of IgA than “true”commensals (Slack et al., 2012, Front. Immun. 3:100). For example, SFBand Helicobacter species are potent inducers IgA responses in theintestine (Umesaki et al., 1999, Infection and Immunity 67:3504-3511;Talham et al., 1999, Infection and Immunity 67:1992-2000). Theinflammasome is a critical component of the innate immune system thatorchestrates the activation of Caspase-1 and release of the inflammatorycytokines IL-1β and IL-18 in response to infection or damage. Micelacking components of the inflammasome, such as the signaling adaptorapoptosis-associated speck-like protein containing a CARD (ASC), harbora dysbiotic microbiota that is colitogenic and can be transmitted towild type mice through co-housing (Strowig et al., 2012, Nature481:278-286). In particular, acquisition of bacteria from the familyPrevotellaceae has been implicated in colitis development in dysbioticmice (Elinav et al., 2011, Cell 145:745-757).

Despite considerable effort, the identification of specific pathobiontsresponsible for driving the development of disease in humans has provendifficult due to the complexity and diversity of the microbiota, as wellas the influence of host genetics and environment on diseasesusceptibility. Therefore, simple metagenomic studies comparing themicrobiota of diseased and normal individuals may fail to identifydisease-causing bacteria because these bacteria may be present in bothgroups, but only cause disease in a subset of susceptible individuals.

There is a need in the art to identify bacteria in the microbiota of asubject that can lead to the development or progression of diseases anddisorders in the subject. The present invention addresses this unmetneed.

SUMMARY OF THE INVENTION

The invention relates to the discovery that secretory antibodies can beused to detect and identify microbes present in the microbiota of asubject that influence susceptibility to or contribute to thedevelopment or progression of diseases or disorders, includinginflammatory diseases and disorders. In one embodiment, the invention isa method of identifying a type of bacteria in the microbiota of asubject that contributes to the development or progression of aninflammatory disease or disorder in the subject, including the steps of:isolating secretory antibody-bound bacteria from the subject'sbiological sample, amplifying bacterial nucleic acid from secretoryantibody-bound bacteria so isolated, determining the sequences of theamplicons, identifying the type of antibody-bound bacteria present inthe subject's biological sample by identifying nucleic acid sequencesthat are indicative of particular types of bacteria. In someembodiments, the microbiota of the subject is on or near mucosal surfaceof the subject selected from the group consisting of thegastrointestinal tract, the respiratory tract, genitourinary tract andmammary gland. In some embodiments, the biological sample is at leastone of a fecal sample, a mucus sample, a sputum sample, and a breastmilk sample. In some embodiments, the bacterial nucleic acid is 16SrRNA. In some embodiments, the secretory antibody is at least oneselected from the group consisting of IgA1, IgA2, and IgM. In someembodiments, the inflammatory disease or disorder is at least oneinflammatory disease or disorder selected from the group consisting ofinflammatory bowel disease, celiac disease, colitis, intestinalhyperplasia, metabolic syndrome, obesity, rheumatoid arthritis, liverdisease, hepatic steatosis, fatty liver disease, non-alcoholic fattyliver disease (NAFLD), and non-alcoholic steatohepatitis (NASH). In someembodiments, the subject is human.

In another embodiment, the invention is a method of diagnosing aninflammatory disease or disorder in a subject in need thereof byidentifying a type of bacteria in the microbiota of the subject thatcontributes to the development or progression of an inflammatory diseaseor disorder, including the steps of: isolating secretory antibody-boundbacteria from the subject's biological sample, amplifying bacterialnucleic acid from secretory antibody-bound bacteria so isolated,determining the sequences of the amplicons so amplified, and identifyingthe type of antibody-bound bacteria present in the subject's biologicalsample by identifying nucleic acid sequences that are indicative ofparticular types of bacteria, wherein when the type of antibody-boundbacteria present in the subject's biological sample is a type ofbacteria that contributes to the development or progression of aninflammatory disease or disorder, the subject is diagnosed with theinflammatory disease or disorder. In some embodiments, the microbiota ofthe subject is on or near mucosal surface of the subject selected fromthe group consisting of the gastrointestinal tract, the respiratorytract, genitourinary tract and mammary gland. In some embodiments, thebiological sample is at least one of a fecal sample, a mucus sample, asputum sample, and a breast milk sample. In some embodiments, thebacterial nucleic acid is 16S rRNA. In some embodiments, the secretoryantibody is at least one selected from the group consisting of IgA1,IgA2, and IgM. In some embodiments, the inflammatory disease or disorderis at least one inflammatory disease or disorder selected from the groupconsisting of inflammatory bowel disease, celiac disease, colitis,intestinal hyperplasia, metabolic syndrome, obesity, rheumatoidarthritis, liver disease, hepatic steatosis, fatty liver disease,non-alcoholic fatty liver disease (NAFLD), and non-alcoholicsteatohepatitis (NASH). In some embodiments, the subject is human.

In one embodiment, the invention is a method of treating an inflammatorydisease or disorder associated with a secretory antibody-bound bacteriain the microbiota of a subject in need thereof, the method comprisingadministering to the subject at least one therapy to diminish the numberof at least one type of bacteria that is over-represented in themicrobiota of the subject. In some embodiments, the at least one therapyis at least one selected from the group consisting of at least onevaccine, at least one antibiotic, and at least one passiveimmunotherapy. In some embodiments, the microbiota of the subject is onor near mucosal surface of the subject selected from the groupconsisting of the gastrointestinal tract, the respiratory tract,genitourinary tract and mammary gland. In some embodiments, thebiological sample is at least one of a fecal sample, a mucus sample, asputum sample, and a breast milk sample. In some embodiments, thesecretory antibody is at least one selected from the group consisting ofIgA1, IgA2, and IgM. In some embodiments, the inflammatory disease ordisorder is at least one inflammatory disease or disorder selected fromthe group consisting of inflammatory bowel disease, celiac disease,colitis, intestinal hyperplasia, metabolic syndrome, obesity, rheumatoidarthritis, liver disease, hepatic steatosis, fatty liver disease,non-alcoholic fatty liver disease (NAFLD), and non-alcoholicsteatohepatitis (NASH). In some embodiments, the subject is human. Insome embodiments, the therapy induces an immune response directedagainst at least one type of secretory antibody-bound bacteria presentin the microbiota of the subject. In some embodiments, the methodfurther comprises administering to the subject at least one probiotic toincrease the number of at least one type of bacteria under-representedin the microbiota of the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1, comprising FIGS. 1A-1C, depicts the results of experimentsdemonstrating that IgA coating is uneven across microbial taxa. (FIG.1A) Overview of IgA-based cell sorting of fecal bacteria combined with16S rRNA sequencing (IgA-SEQ). (FIG. 1B) Representative cell sorting ofIgA+ fecal bacteria from mice. A cartoon is used to illustrateseparation of IgA+ and IgA− bacteria from total fecal bacteria. (FIG.1C) Principal Coordinates Analysis of weighted UniFrac distances ofPresort (total fecal bacteria), IgA+, IgA−, and mock-sorted (MACS andFACS) samples. PC, Principal Coordinate. PERMANOVA comparisons ofweighted UniFrac distances of Presort, IgA+, IgA−, and mock-sortedsamples demonstrated that IgA+ bacteria were phylogenetically distinctfrom Presort and IgA− fractions (P<0.05), while IgA− bacteria also werenot significantly different from total bacteria (P=0.266). Mock sortingdid not significantly alter the observed phylogenetic composition offecal bacteria (Presort versus MACS: P=0.655; Presort versus FACS:P=0.606). Mock-sorted samples were stained with anti-IgA and sorted byMACS before mixing positive and negative fractions and FACS sorting oftotal bacteria.

FIG. 2, comprising FIGS. 2A-2B, depicts the results of experimentsassessing IgA coating of fecal bacteria from Specific Pathogen Free(SPF) and SPF^(dysbiosis) mice. Average relative abundances and IgAcoating indices (ICI) for Total (Presort), IgA+ and IgA− bacterialgenera from (FIG. 2A) C57Bl/6 SPF mice (n=17 samples) and (FIG. 2B)SPF^(dysbiosis) mice (n=14 samples). SPF^(dysbiosis) mice were co-housedwith Asc^(−/−) mice for at least 6 weeks to allow for the acquisition ofdysbiosis. Relative abundance heatmaps are depicted on a logarithmicscale. Genera that are highly coated with IgA (significantly higherrelative abundance in the IgA+ fraction as compared to the IgA− fractionby LEfSe (P<0.05) and Linear Discriminant Analysis Score>2), andWilcoxon rank-sum (P<0.05)) are labeled in red (###), while genera thatshow low or no IgA coating (significantly higher relative abundance inthe IgA− fraction as compared to the IgA+ fraction by LEfSe and Wilcoxonrank-sum) are labeled in blue (##).

FIG. 3, comprising FIGS. 3A-3C, depicts the results of experimentsassessing IgA coating of fecal bacteria from healthy humans andinflammatory bowel disease patients. Depicted in the main heatmap areIgA coating index (ICI) scores for bacterial species from 20 healthyhumans (FIG. 3A), 27 Crohn's disease patients (FIG. 3B), and 8 patientswith UC (FIG. 3C). Each column represents an individual human subject.Bacterial taxa are clustered (complete linkage clustering usingEuclidean distance) based ICI scores observed in healthy humans.Bacterial taxa with significantly higher relative abundance in the IgA+fraction as compared to the IgA− fraction by LEfSe and Wilcoxon rank-sumare considered to be highly coated with IgA and are labeled in red(###). Bacterial taxa with significantly higher relative abundance inthe IgA− fraction as compared to the IgA+ fraction by LEfSe and Wilcoxonrank-sum are considered to show low to no IgA coating and are labeled inblue (##). Bacterial taxa showing no significant difference in abundancein the IgA+ and IgA− fractions are labeled black. The leftmost heatmapsummarizes the statistical comparisons between relative taxonomicabundance in the IgA+ and IgA− negative fraction. IgA coated bacteriaare marked in red (###), low coated bacteria are marked in blue (##).Comparisons between ICI coating scores in Control and IBD patients aresummarized as follows: gray marks no difference between diseased andcontrol, green marks taxa where ICI scores are higher in controls thanin diseased patients, and purple marks taxa where ICI scores aresignificantly lower in controls than in diseased patients. Significancelevels for LEfSe and Wilcoxon rank-sum were P<0.05 and LinearDiscriminant Analysis Score>2, and P<0.05, respectively.

FIG. 4, comprising FIGS. 4A-4B, depicts the results of experimentsassessing IgA coating of fecal bacteria from SPF C57Bl/6 mice. (FIG. 4A)Staining of IgA coated intestinal bacteria from C57Bl/6 SPF andRag2^(−/−) mice, which lack immunoglobulins. (FIG. 4B) Gating onIgA-stained bacteria demonstrates that the vast majority of IgA+ eventsfall within the designated FSC and SSC gate. SSC, side scatter. FSC,forward scatter.

FIG. 5, comprising FIGS. 5A-4C, depicts the results of the sorting ofIgA+ and IgA− fecal bacteria. (FIG. 5A) Post-sort purity of IgA+ andIgA− fractions. (FIG. 5B) IgA concentrations in total, IgA+ and IgA−bacterial fractions (after MACS sorting) as determined by wholebacterial-cell ELISA. (FIG. 5C) Average relative abundances of bacterialgenera of >1% abundance in Presort (Total), IgA+, IgA−, and mock-sorted(MACS and FACS) samples (n=4 mice). UC, unclassified.

FIG. 6, comprising FIGS. 6A-6C, depicts the results of experimentsassessing IgA coating of intestinal bacteria from SPF mice. (FIG. 6A)Principal Coordinate Analysis and PERMANOVA comparisons of weightedUniFrac distances of Total (Presort), IgA+ and IgA− fecal bacteria fromSPF mice (n=17). PC, Principal Coordinate. (FIG. 6B) LEfSe comparisonsof IgA+ and IgA− bacterial genera from SPF mice. (FIG. 6C) Relativeabundance of bacterial families from total, IgA coated (IgA+) andnoncoated (IgA−) intestinal bacteria from individual groups of SPF mice.Depicted are bacteria of >1% abundance as averaged from eight pairs ofseparately housed mice sampled at least two times. Significance levelsfor LEfSe were P<0.05 and Linear Discriminant Analysis Score>2. UC,unclassified.

FIG. 7, comprising FIGS. 7A-7D, depicts the results of experimentsdemonstrating that inflammasome-mediated dysbiosis leads to ahypersensitivity to DSS-induced colitis and increases the level of IgAcoating of the microbiota. (FIG. 7A) Principal Coordinate Analysis ofweighted UniFrac distances from SPF (n=17) and SPF^(dysbiosis) mice(n=14). PERMANOVA P=0.001. (FIG. 7B) Average relative abundance ofbacterial families in the intestinal microbiota from SPF andSPF^(dysbiosis) mice. Prevotellaceae is marked with an arrow. UC,unclassified. (FIG. 7C) Dextran Sodium Sulfate (DSS)-induced colitis inSPF and SPF^(dysbiosis) mice. Mice were treated with 2% DSS ad libitumin the drinking water for 7 days and weight was followed for 14 days.(FIG. 7D) IgA coating of fecal bacteria from SPF and SPF^(dysbiosis)mice as measured by flow cytometry. SPF^(dysbiosis) mice were co-housedwith Asc^(−/−) mice for at least 6 weeks to allow for the acquisition ofdysbiosis. ***P<0.001 (unpaired Student's t-test).

FIG. 8, comprising FIGS. 8A-8C, depicts the results of experimentsassessing IgA coating of intestinal bacteria from SPF^(dysbiosis) mice.(FIG. 8A) Principal Coordinate Analysis and PERMANOVA comparisons ofweighted UniFrac distances of Total (Presort), IgA+ and IgA− fecalbacteria from SPF (n=17) and SPF^(dysbiosis) mice (n=14). PC, PrincipalCoordinate. (FIG. 8B) LEfSe comparisons of IgA+ and IgA− bacterialgenera from SPF^(dysbiosis) mice. (FIG. 8C) Relative abundance ofbacterial families in total (Total), IgA coated (IgA+), and uncoated(IgA−) intestinal bacteria in SPF^(dysbiosis) mice. Six pairs ofseparately housed dysbiotic mice (A-F) were sampled at least two timesand the average of the two measurements is shown. Depicted are bacteriaof >1% abundance. Significance levels for LEfSe were P<0.05 and LinearDiscriminant Analysis Score>2. UC, unclassified.

FIG. 9 depicts the results of experiments assessing IgA coating ofluminal and mucus-associated bacteria from the small intestine, cecumand colon of SPF^(dysbiosis) mice. Relative abundances of luminal andmucus-associated bacteria from the small intestine, cecum and colon ofSPF^(dysbiosis) mice (n=5).

FIG. 10 depicts the results of experiments assessing IgA coating offecal bacteria from healthy, Crohn's disease and Ulcerative colitispatients. IgA coating of fecal bacteria from 20 healthy subjects, 27Crohn's disease patients (CD) and 8 Ulcerative colitis patients (UC) asmeasured by flow cytometry. *P<0.05, ***P<0.001 (one-way ANOVA).

FIG. 11, comprising FIGS. 11A-11C, depicts relative abundance heatmapsof intestinal bacteria from healthy humans, Crohn's disease patients,and patients with Ulcerative colitis. Relative abundance is depicted ona log scale to allow for visualization of low abundance taxa. Bacterialtaxa are clustered (complete linkage clustering using Euclideandistance) based ICI scores observed in healthy humans. As in FIG. 3,bacterial taxa with significantly higher relative abundance in the IgA+fraction as compared to the IgA− fraction by LEfSe and Wilcoxon rank-sumare considered to be highly coated with IgA and are labeled in red(###). Bacterial taxa with significantly higher relative abundance inthe IgA− fraction as compared to the IgA+ fraction by LEfSe and Wilcoxonrank-sum are considered to show low to no IgA coating and are labeled inblue (##). Bacterial taxa showing no significant difference in abundancein the IgA+ and IgA− fractions are labeled black. Each column representsan individual human subject.

FIG. 12, comprising FIGS. 12A-12D, depicts the distributions of IgAcoating index (ICI) scores in healthy humans and patients with Crohn'sdisease or Ulcerative colitis. (FIG. 12A-12C) Depicted are the number ofIgA coated bacteria with an ICI score of >2, >10, and >100 in healthyhumans and Crohn's disease or Ulcerative colitis patients. (FIG. 12D)Average ICI score per person.

FIG. 13, comprising FIG. 13A-13C, depicts LEfSe comparisons of relativeabundance of bacterial taxa from IgA+ and IgA− fractions of fecalbacteria from healthy humans and patients with Crohn's disease orUlcerative Colitis. Bacterial taxa with significantly higher relativeabundance in the IgA+ fraction as compared to the IgA− fraction by LEfSeare shown in green. Bacterial taxa with significantly higher relativeabundance in the IgA− fraction as compared to the IgA+ fraction by LEfSeare shown in red. Bacterial taxa showing no significant difference inabundance in the IgA+ and IgA− fractions are omitted from these graphs.Significance levels for LEfSe were as follows: Kruskal-Wallis sum-rankP<0.05, Wilcoxon rank-sum P<0.05 and Linear Discriminant AnalysisScore>2.

FIG. 14 depicts the results of experiments assessing the IgA coating offecal bacteria from healthy and obese adolescents. Depicted in the mainheatmap are IgA coating index (ICI) scores for bacterial species from 4healthy and 15 obese adolescents. Each column represents an individualhuman subject. Bacterial taxa are clustered (complete linkage clusteringusing Euclidean distance) based ICI scores. Bacterial taxa from obesepatients with significantly higher relative abundance in the IgA+fraction as compared to the IgA− fraction by LEfSe are considered to behighly coated with IgA and are labeled in red (###).

DETAILED DESCRIPTION

The present invention relates to the discovery that secretory antibodiescan be used to detect and identify microbes present in the microbiota ofa subject that influence susceptibility to or contribute to thedevelopment or progression of diseases or disorders. Described hereinare novel methods that combine flow cytometry-based microbial cellsorting and genetic analyses to detect, to isolate and to identifysecretory antibody-coated (e.g., IgA-coated) microbes from themicrobiota of a subject. Because disease-causing members of themicrobiota, including pathobionts, are recognized by the subject'simmune system, their presence triggers an immune response, includingantibody production and secretion. In some embodiments of the methodsdescribed herein, the presence of an immune response (e.g., antibodyproduction and secretion) in the subject serves as a marker and a meansfor isolating and identifying pathobionts, and putative pathobionts,that are the targets of the subject's immune response. Thus, the methodsdescribed herein can isolate and identify microbes present in themicrobiota of a subject that influence susceptibility to or contributeto the development or progression of a disease or disorder. Themicrobiota of the subject can be any microbiota present on any mucosalsurface of subject where antibody is secreted, including thegastrointestinal tract, the respiratory tract, genitourinary tract andmammary gland.

In various embodiments, the present invention relates to the isolationand identification of members of the microbiota that influence thedevelopment and progression of a disease or disorder, such as aninflammatory disease or disorder. Thus, the invention relates tocompositions and methods for detecting and determining the identity ofsecretory antibody-coated constituents of a subject's microbiota todetermine whether the secretory antibody-coated constituents of asubject's microbiota contribute to an altered microbiota associated withan inflammatory disease or disorder. In various embodiments, therelative proportions of the secretory antibody-coated and uncoatedconstituents of a subject's microbiota are indicative of an alteredmicrobiota associated with an inflammatory disease or disorder. In someembodiments, the detection and identification of secretoryantibody-coated constituents of the microbiota of the subject are usedto diagnose the subject as having, or as at risk of developing, aninflammatory disease or disorder. Thus, in some embodiments, the alteredmicrobiota of a subject influences susceptibility to or contributes tothe development or progression of a disease or disorder, such as aninflammatory disease or disorder. In various embodiments, theinflammatory diseases and disorders associated with altered microbiotahaving secretory antibody-coated constituents include, but are notlimited to, at least one of: inflammatory bowel disease, celiac disease,colitis, intestinal hyperplasia, metabolic syndrome, obesity, rheumatoidarthritis, liver disease, hepatic steatosis, fatty liver disease,non-alcoholic fatty liver disease (NAFLD), or non-alcoholicsteatohepatitis (NASH).

Further, the present invention relates to methods of modifying analtered microbiota having secretory antibody-coated constituents in asubject in need thereof. In some embodiments, the invention providescompositions and methods for supplementing constituents of an alteredmicrobiota that are under-represented in the altered microbiota, ascompared with a normal microbiota, to restore the subject's microbiotato a normal microbiota. In other embodiments, the invention providescompositions and methods for diminishing constituents of an alteredmicrobiota that are over-represented in the altered microbiota ascompared with a normal microbiota, such as over-represented secretoryantibody-coated constituents, to restore the subject's microbiota to anormal microbiota. In further embodiments, the invention providescompositions and methods for both supplementing constituents of analtered microbiota that are under-represented in the altered microbiota,as well as diminishing constituents of an altered microbiota that areover-represented in the altered microbiota, as compared with a normalmicrobiota, to restore the subject's microbiota to a normal microbiota.

As used throughout herein, constituents of an altered microbiota thatare over-represented in the altered microbiota as compared with a normalmicrobiota, include constituents that are uniquely present in thealtered microbiota as compared with a normal microbiota.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

A disease or disorder is “alleviated” if the severity of a sign orsymptom of the disease or disorder, the frequency with which such a signor symptom is experienced by a patient, or both, is reduced.

The term “dysbiosis,” as used herein, refers to imbalances in quality,absolute quantity, or relative quantity of members of the microbiota ofa subject, which is sometimes, but not necessarily, associated with thedevelopment or progression of a disease or disorder.

The term “microbiota,” as used herein, refers to the population ofmicroorganisms present within or upon a subject. The microbiota of asubject includes commensal microorganisms found in the absence ofdisease and may also include pathobionts and disease-causingmicroorganisms found in subjects with or without a disease or disorder.

The term “pathobiont,” as used herein, refers to potentiallydisease-causing members of the microbioata that are present in themicrobiota of a non-diseased or a diseased subject, and which has thepotential to contribute to the development or progression of a diseaseor disorder.

An “effective amount” or “therapeutically effective amount” of acompound is that amount of a compound which is sufficient to provide abeneficial effect to the subject to which the compound is administered.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a compound, composition, or methodof the invention in a kit. The instructional material of the kit of theinvention can, for example, be affixed to a container which contains theidentified compound, composition, or method of the invention or beshipped together with a container which contains the identifiedcompound, composition, or method of the invention. Alternatively, theinstructional material can be shipped separately from the container withthe intention that the instructional material and the compound,composition, or method of the invention be used cooperatively by therecipient.

The term “microarray” refers broadly to both “DNA microarrays” and “DNAchip(s),” and encompasses all art-recognized solid supports, and allart-recognized methods for affixing nucleic acid molecules thereto orfor synthesis of nucleic acids thereon.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in vivo, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis, by way of non-limiting examples, a human, a dog, a cat, a horse, orother domestic mammal.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs or symptoms of pathology, for the purpose of diminishingor eliminating those signs or symptoms.

As used herein, “treating a disease or disorder” means reducing theseverity and/or frequency with which a sign or symptom of the disease ordisorder is experienced by a patient. Disease and disorder are usedinterchangeably herein.

The phrase “biological sample” as used herein, is intended to includeany sample comprising a cell, a tissue, feces, or a bodily fluid inwhich the presence of a microbe, nucleic acid or polypeptide is presentor can be detected. Samples that are liquid in nature are referred toherein as “bodily fluids.” Biological samples may be obtained from apatient by a variety of techniques including, for example, by scrapingor swabbing an area of the subject or by using a needle to obtain bodilyfluids. Methods for collecting various body samples are well known inthe art.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, intracellular antibodies(“intrabodies”), Fv, Fab and F(ab)2, as well as single chain antibodies(scFv), heavy chain antibodies, such as camelid antibodies, andhumanized antibodies (Harlow et al., 1999, Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow etal., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.;Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird etal., 1988, Science 242:423-426).

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

As used herein, the term “heavy chain antibody” or “heavy chainantibodies” comprises immunoglobulin molecules derived from camelidspecies, either by immunization with a peptide and subsequent isolationof sera, or by the cloning and expression of nucleic acid sequencesencoding such antibodies. The term “heavy chain antibody” or “heavychain antibodies” further encompasses immunoglobulin molecules isolatedfrom an animal with heavy chain disease, or prepared by the cloning andexpression of VH (variable heavy chain immunoglobulin) genes from ananimal.

As used herein, an “immunoassay” refers to any binding assay that usesan antibody capable of binding specifically to a target molecule todetect and quantify the target molecule.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes and binds to a specificantigen, but does not substantially recognize or bind other molecules ina sample. For example, an antibody that specifically binds to an antigenfrom one species may also bind to that antigen from one or more species.But, such cross-species reactivity does not itself alter theclassification of an antibody as specific. In another example, anantibody that specifically binds to an antigen may also bind todifferent allelic forms of the antigen. However, such cross reactivitydoes not itself alter the classification of an antibody as specific.

In some instances, the terms “specific binding” or “specificallybinding,” can be used in reference to the interaction of an antibody, aprotein, or a peptide with a second chemical species, to mean that theinteraction is dependent upon the presence of a particular structure(e.g., an antigenic determinant or epitope) on the chemical species; forexample, an antibody recognizes and binds to a specific proteinstructure rather than to proteins generally.

A “coding region” of a gene consists of the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene which are homologous with or complementary to, respectively,the coding region of an mRNA molecule which is produced by transcriptionof the gene.

A “coding region” of a mRNA molecule also consists of the nucleotideresidues of the mRNA molecule which are matched with an anti-codonregion of a transfer RNA molecule during translation of the mRNAmolecule or which encode a stop codon. The coding region may thusinclude nucleotide residues comprising codons for amino acid residueswhich are not present in the mature protein encoded by the mRNA molecule(e.g., amino acid residues in a protein export signal sequence).

“Complementary” as used herein to refer to a nucleic acid, refers to thebroad concept of sequence complementarity between regions of two nucleicacid strands or between two regions of the same nucleic acid strand. Itis known that an adenine residue of a first nucleic acid region iscapable of forming specific hydrogen bonds (“base pairing”) with aresidue of a second nucleic acid region which is antiparallel to thefirst region if the residue is thymine or uracil. Similarly, it is knownthat a cytosine residue of a first nucleic acid strand is capable ofbase pairing with a residue of a second nucleic acid strand which isantiparallel to the first strand if the residue is guanine. A firstregion of a nucleic acid is complementary to a second region of the sameor a different nucleic acid if, when the two regions are arranged in anantiparallel fashion, at least one nucleotide residue of the firstregion is capable of base pairing with a residue of the second region.Preferably, the first region comprises a first portion and the secondregion comprises a second portion, whereby, when the first and secondportions are arranged in an antiparallel fashion, at least about 50%,and preferably at least about 75%, at least about 90%, or at least about95% of the nucleotide residues of the first portion are capable of basepairing with nucleotide residues in the second portion. More preferably,all nucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in its normal context in aliving animal is not “isolated,” but the same nucleic acid or peptidepartially or completely separated from the coexisting materials of itsnatural context is “isolated.” An isolated nucleic acid or protein canexist in substantially purified form, or can exist in a non-nativeenvironment such as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “probiotic” refers to one or more bacteria that can beadministered to a subject to aid in the restoration of a subject'smicrobiota by increasing the number of bacteria that areunder-represented in the subject's microbiota.

“Variant” as the term is used herein, is a nucleic acid sequence or apeptide sequence that differs in sequence from a reference nucleic acidsequence or peptide sequence respectively, but retains essentialbiological properties of the reference molecule. Changes in the sequenceof a nucleic acid variant may not alter the amino acid sequence of apeptide encoded by the reference nucleic acid, or may result in aminoacid substitutions, additions, deletions, fusions and truncations.Changes in the sequence of peptide variants are typically limited orconservative, so that the sequences of the reference peptide and thevariant are closely similar overall and, in many regions, identical. Avariant and reference peptide can differ in amino acid sequence by oneor more substitutions, additions, deletions in any combination. Avariant of a nucleic acid or peptide can be a naturally occurring suchas an allelic variant, or can be a variant that is not known to occurnaturally. Non-naturally occurring variants of nucleic acids andpeptides may be made by mutagenesis techniques or by direct synthesis.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The present invention relates to the discovery that secretoryantibodies, such as IgA1, IgA2 or IgM, can be used to detect andidentify microbes present in the microbiota of a subject that influencesusceptibility to or contribute to the development or progression of adiseases or disorder, such as an inflammatory disease or disorder. Thus,the invention relates to compositions and methods for detecting,identifying and determining the absolute number or relative proportionsof the secretory antibody-coated and uncoated constituents of asubject's microbiota, to determine whether a subject's microbiota is analtered microbiota associated with a disease or disorder, such as aninflammatory disease or disorder. Further, the present invention relatesto methods of modifying an altered microbiota population in a subject inneed thereof. The microbiota of the subject can be any microbiotapresent on any mucosal surface of subject where antibody is secreted,including the gastrointestinal tract, the respiratory tract,genitourinary tract and mammary gland.

Methods of Identifying

The methods of the invention are useful for detecting, identifying anddetermining the absolute number or relative proportions of secretoryantibody-coated and uncoated constituents of a subject's microbiota, todetermine whether a subject's microbiota is an altered microbiotaassociated with a disease or disorder, such as an inflammatory diseaseor disorder. In some embodiments, the methods of the invention combine aflow cytometry-based microbial cell sorting and genetic analyses todetect, to isolate and to identify secretory antibody-coated microbesfrom the microbiota of a subject. Pathobionts, as well as otherdisease-causing microbes, present in the microbiota of the of thesubject are recognized by the subject's immune system, which triggers animmune response, including antibody production and secretion, directedagainst the pathobionts, and disease-causing microbes. Thus, in someembodiments of the methods of the invention, specifically bindingsecretory antibodies (e.g., IgA, IgM) produced by the subject andsecreted through the mucosa of the subject, serve as a marker and ameans for isolating and identifying putative pathobionts, pathobionts,and other disease-causing bacteria, that are the targets of thesubject's immune response. In various embodiments of the methods of theinvention, the secretory antibody is IgA (i.e., IgA1, IgA2), or IgM, orany combination thereof. The microbiota of the subject can be anymicrobiota present on any mucosal surface of subject where antibody issecreted, including the gastrointestinal tract, the respiratory tract,genitourinary tract and mammary gland.

In various embodiments, the present invention relates to the isolationand identification of constituents of the microbiota of a subject thatinfluence the development and progression of a disease or disorder, suchas an inflammatory disease and disorder. In one embodiment, theinvention relates to compositions and methods for detecting anddetermining the identity of secretory antibody-coated constituents of asubject's microbiota to determine whether the secretory antibody-coatedconstituents of a subject's microbiota form an altered microbiotaassociated with an inflammatory disease or disorder. In variousembodiments, the relative proportions of the secretory antibody-coatedand uncoated constituents of a subject's microbiota are indicative of analtered microbiota associated with an inflammatory disease or disorder.In some embodiments, the detection and identification of secretoryantibody-coated constituents of the microbiota of the subject are usedto diagnose the subject as having, or as at risk of developing, aninflammatory disease or disorder. In other embodiments, the detectionand identification of secretory antibody-coated constituents of themicrobiota of the subject are used to diagnose the subject as having, oras at risk of developing, a recurrence or flare of an inflammatorydisease or disorder. In other embodiments, the detection andidentification of secretory antibody-coated constituents of themicrobiota of the subject are used to diagnose the subject as having, oras likely to have, remission or an inflammatory disease or disorder. Invarious embodiments, the inflammatory diseases and disorders associatedwith altered microbiota having secretory antibody-coated constituentsinclude, but are not limited to, at least one of: inflammatory boweldisease, celiac disease, colitis, intestinal hyperplasia, metabolicsyndrome, obesity, rheumatoid arthritis, liver disease, hepaticsteatosis, fatty liver disease, non-alcoholic fatty liver disease(NAFLD), or non-alcoholic steatohepatitis (NASH).

In one embodiment, the invention is a method for determining therelative proportions of the types of secretory antibody-coatedconstituents of a subject's microbiota, to identify constituents of asubject's microbiota that are associated with the development orprogression of an inflammatory disease or disorder. In some embodiments,the detection of particular types of secretory antibody-coatedconstituents of the subject's microbiota is used to diagnose the subjectas having, or as at risk of developing, an inflammatory disease ordisorder. In various embodiments, the inflammatory disease or disorderassociated with secretory antibody-coated constituents of the subject'smicrobiota include, but are not limited to, at least one of:inflammatory bowel disease, celiac disease, colitis, intestinalhyperplasia, metabolic syndrome, obesity, rheumatoid arthritis, liverdisease, hepatic steatosis, fatty liver disease, non-alcoholic fattyliver disease (NAFLD), and non-alcoholic steatohepatitis (NASH). In someembodiments, the secretory antibody-coated constituent of the subject'smicrobiota associated with the develop or progression of an inflammatorydisease or disorder in the subject is a Segmented Filamentous Bacteria(SFB) or Helicobacter flexispira. In other embodiments, the secretoryantibody-coated constituent of the subject's microbiota associated withthe development or progression of an inflammatory disease or disorder inthe subject is a bacteria from a family selected from the groupconsisting of Lactobacillus, Helicobacter, S24-7, Erysipelotrichaceaeand Prevotellaceae. In some embodiments, the bacteria from the familyPrevotellaceae is a bacteria from the genera of Paraprevotella orPrevotella. In other embodiments, the secretory antibody-coatedconstituent of the subject's microbiota associated with the developmentor progression of an inflammatory disease or disorder in the subject isat least one selected from Eubacterium, Eubacterium biforme, Eubacteriumdolichum, Ruminococcus gnavus, Acidaminococcus, Actinomyces,Allobaculum, Anaerostipes, Bacteroides, Bacteroides fragilis,Bacteroides Other, Bifidobacterium, Bifidobacterium adolescentis,Bifidobacterium Other, Blautia, Blautia obeum, Blautia Other, Blautiaproducta, Bulleidia Other, Clostridium, Clostridium perfringens,Collinsella aerofaciens, Coprococcus, Coprococcus catus, Dialister,Eggerthella lenta, Erysipelotrichaceae, Faecalibacterium prausnitzii,Haemophilus parainfluenzae, Lachnospiraceae, Lachnospiraceae other,Lactobacillus, Lactobacillus mucosae, Lactobacillus Other, Lactobacillusreuteri, Lactobacillus zeae, Oscillospira, Pediococcus Other,Rikenellaceae, Roseburia, Roseburia faecis, Ruminococcaceae,Ruminococcus, Ruminococcus bromii, SMB53, Streptococcus, Streptococcusluteciae, Streptococcus Other, Sutterella, Turicibacter, UCClostridiales, UC Erysipelotrichaceae, UC Ruminococcaceae, Veillonella,Veillonella dispar, and Weissella.

In some embodiments, the invention is a method of identifying the typeor types of secretory antibody-bound bacteria present in the microbiotaof a subject that contribute to the development or progression of aninflammatory disease or disorder in the subject. In other embodiments,the invention is a method of diagnosing an inflammatory disease ordisorder in a subject by identifying a type or types of secretoryantibody-bound bacteria in the microbiota of the subject that contributeto the development or progression of an inflammatory disease ordisorder.

Specific alterations in a subject's microbiota, including the presenceof secretory antibody-coated constituents, can be detected using variousmethods, including without limitation quantitative PCR orhigh-throughput sequencing methods which detect relative proportions ofmicrobial genetic markers in a total heterogeneous microbial population.In some embodiments, the microbial genetic marker is a bacterial geneticmarker. In particular embodiments, the bacterial genetic marker is atleast some portion of the 16S rRNA. In some embodiments, the relativeproportion of particular constituent bacterial phyla, classes, orders,families, genera, and species present in the microbiota of a subject isdetermined. In other embodiments, the relative proportion of secretoryantibody-coated and/or uncoated constituent bacterial phyla, classes,orders, families, genera, and species present in the microbiota of asubject is determined. In some embodiments, the relative proportion ofparticular constituent bacterial phyla, classes, orders, families,genera, and species present in the microbiota of a subject is determinedand compared with that of a comparator normal microbiota. In otherembodiments, the relative proportion of secretory antibody-coated and/oruncoated constituent bacterial phyla, classes, orders, families, genera,and species present in the microbiota of a subject is determined andcompared with that of a comparator normal microbiota. In variousembodiments, the comparator normal microbiota is, by way of non-limitingexamples, a microbiota of a subject known to be free of an inflammatorydisorder, or a historical norm, or a typical microbiota of thepopulation of which the subject is a member.

In one embodiment, the method of the invention is a diagnostic assay fordiagnosing an inflammatory disease or disorder associated with analtered microbiota in a subject in need thereof, by determining theabsolute or relative abundance of particular types of secretoryantibody-coated constituents of the subject's microbiota present in abiological sample derived from the subject. In some embodiments, thesubject is diagnosed as having an inflammatory disease or disorder whenparticular types of secretory antibody-coated bacteria are determined tobe present in the biological sample derived from the subject withincreased relative abundance. In some embodiments, the secretoryantibody-coated bacteria determined to be present in the biologicalsample derived from the subject with increased relative abundance is aSegmented Filamentous Bacteria (SFB) or Helicobacter flexispira. In someembodiments, the secretory antibody-coated bacteria determined to bepresent in the biological sample derived from the subject with increasedrelative abundance is a bacteria from a family selected from the groupconsisting of Lactobacillus, Helicobacter, S24-7, Erysipelotrichaceaeand Prevotellaceae. In some embodiments, the bacteria from the familyPrevotellaceae is a bacteria from the genera of Paraprevotella orPrevotella. In other embodiments, the secretory antibody-coatedconstituent of the subject's microbiota associated with the developmentor progression of an inflammatory disease or disorder in the subject isat least one selected from Eubacterium, Eubacterium biforme, Eubacteriumdolichum, Ruminococcus gnavus, Acidaminococcus, Actinomyces,Allobaculum, Anaerostipes, Bacteroides, Bacteroides fragilis,Bacteroides Other, Bifidobacterium, Bifidobacterium adolescentis,Bifidobacterium Other, Blautia, Blautia obeum, Blautia Other, Blautiaproducta, Bulleidia Other, Clostridium, Clostridium perfringens,Collinsella aerofaciens, Coprococcus, Coprococcus catus, Dialister,Eggerthella lenta, Erysipelotrichaceae, Faecalibacterium prausnitzii,Haemophilus parainfluenzae, Lachnospiraceae, Lachnospiraceae other,Lactobacillus, Lactobacillus mucosae, Lactobacillus Other, Lactobacillusreuteri, Lactobacillus zeae, Oscillospira, Pediococcus Other,Rikenellaceae, Roseburia, Roseburia faecis, Ruminococcaceae,Ruminococcus, Ruminococcus bromii, SMB53, Streptococcus, Streptococcusluteciae, Streptococcus Other, Sutterella, Turicibacter, UCClostridiales, UC Erysipelotrichaceae, UC Ruminococcaceae, Veillonella,Veillonella dispar, and Weissella.

The results of the diagnostic assay can be used alone, or in combinationwith other information from the subject, or from the biological samplederived from the subject.

In the assay methods of the invention, a test biological sample from asubject is assessed for the absolute or relative abundance of secretoryantibody-coated and uncoated constituents of the microbiota. The testbiological sample can be an in vitro sample or an in vivo sample. Invarious embodiments, the subject is a human subject, and may be of anyrace, sex and age. Representative subjects include those who aresuspected of having an altered microbiota associated with aninflammatory disease or disorder, those who have been diagnosed with analtered microbiota associated with an inflammatory disease or disorder,those whose have an altered microbiota associated with an inflammatorydisease or disorder, those who have had an altered microbiota associatedwith an inflammatory disease or disorder, those who at risk of arecurrence of an altered microbiota associated with an inflammatorydisease or disorder, those who at risk of a flare of an alteredmicrobiota associated with an inflammatory disease or disorder, andthose who are at risk of developing an altered microbiota associatedwith an inflammatory disease or disorder.

In some embodiments, the test sample is prepared from a biologicalsample obtained from the subject. In some instances, a heterogeneouspopulation of microbes will be present in the biological samples.Enrichment of a microbial population for microbes (e.g., bacteria) boundby secretory antibody (e.g., IgA, IgM) may be accomplished usingseparation technique. For example, microbes of interest may be enrichedby separation the microbes of interest from the initial population usingaffinity separation techniques. Techniques for affinity separation mayinclude magnetic separation using magnetic beads conjugated with anaffinity reagent, affinity chromatography, “panning” with an affinityreagent attached to a solid matrix, e.g. plate, or other convenienttechnique. Other techniques providing separation include fluorescenceactivated cell sorting, which can have varying degrees ofsophistication, such as multiple color channels, low angle and obtuselight scattering detecting channels, impedance channels, etc. Oneexample of an affinity reagent useful in the methods of the invention isan antibody, such as anti-species antibody or anti-isotype (e.g.,anti-IgA, anti-IgM) antibody. The details of the preparation of suchantibodies and their suitability for use as affinity reagents arewell-known to those skilled in the art. In some embodiments, labeledantibodies are used as affinity reagents. Conveniently, these antibodiesare conjugated with a label for use in separation. Labels includemagnetic beads, which allow for direct separation; biotin, which can beremoved with avidin or streptavidin bound to a support; fluorochromes,which can be used with a fluorescence activated cell sorter; or thelike, to allow for ease of separation of the particular cell type.

In various embodiments, the initial population of microbes is contactedwith one or more affinity reagent(s) and incubated for a period of timesufficient to permit the affinity reagent to specifically bind to itstarget. The microbes in the contacted population that become labeled bythe affinity reagent are selected for by any convenient affinityseparation technique, e.g. as described elsewhere herein or as known inthe art. Compositions highly enriched for a microbe of interest (e.g.,secretory antibody-bound bacteria) are achieved in this manner. Theaffinity enriched microbes will be about 70%, about 75%, about 80%,about 85% about 90%, about 95% or more of the composition. In otherwords, the enriched composition can be a substantially pure compositionof the microbes of interest.

In one embodiment, the test sample is a sample containing at least afragment of a bacterial nucleic acid. The term, “fragment,” as usedherein, indicates that the portion of a nucleic acid (e.g., DNA, RNA)that is sufficient to identify it as comprising a bacterial nucleicacid.

In some embodiments, the test sample is prepared from a biologicalsample obtained from the subject. The biological sample can be a samplefrom any source which contains a bacterial nucleic acid (e.g., DNA,RNA), such as a bodily fluid or fecal sample, or a combination thereof.A biological sample can be obtained by any suitable method. In someembodiments, a biological sample containing bacterial DNA is used. Inother embodiments, a biological sample containing bacterial RNA is used.The biological sample can be used as the test sample; alternatively, thebiological sample can be processed to enhance access to nucleic acids,or copies of nucleic acids, and the processed biological sample can thenbe used as the test sample. For example, in various embodiments, nucleicacid is prepared from a biological sample, for use in the methods.Alternatively or in addition, if desired, an amplification method can beused to amplify nucleic acids comprising all or a fragment of an RNA orDNA in a biological sample, for use as the test sample in the assessmentof the presence, absence and proportion of particular types of bacteriapresent in the sample.

In some embodiments, hybridization methods, such as Southern analysis,Northern analysis, or in situ hybridizations, can be used (see CurrentProtocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley &Sons, including all supplements). For example, the presence of nucleicacid from a particular type of bacteria can be determined byhybridization of nucleic acid to a nucleic acid probe. A “nucleic acidprobe,” as used herein, can be a DNA probe or an RNA probe.

The nucleic acid probe can be, for example, a full-length nucleic acidmolecule, or a portion thereof, such as an oligonucleotide of at least15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to appropriate targetRNA or DNA. The hybridization sample is maintained under conditionswhich are sufficient to allow specific hybridization of the nucleic acidprobe to RNA or DNA. Specific hybridization can be performed under highstringency conditions or moderate stringency conditions, as appropriate.In a preferred embodiment, the hybridization conditions for specifichybridization are high stringency. More than one nucleic acid probe canalso be used concurrently in this method. Specific hybridization of anyone of the nucleic acid probes is indicative of the presence of theparticular type of bacteria of interest, as described herein.

In Northern analysis (see Current Protocols in Molecular Biology,Ausubel, F. et al., eds., John Wiley & Sons, supra), the hybridizationmethods described above are used to identify the presence of a sequenceof interest in an RNA, such as unprocessed, partially processed or fullyprocessed rRNA. For Northern analysis, a test sample comprising RNA isprepared from a biological sample from the subject by appropriate means.Specific hybridization of a nucleic acid probe, as described above, toRNA from the biological sample is indicative of the presence of theparticular type of bacteria of interest, as described herein.

Alternatively, a peptide nucleic acid (PNA) probe can be used instead ofa nucleic acid probe in the hybridization methods described herein. PNAis a DNA mimic having a peptide-like, inorganic backbone, such asN-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U)attached to the glycine nitrogen via a methylene carbonyl linker (see,for example, 1994, Nielsen et al., Bioconjugate Chemistry 5:1). The PNAprobe can be designed to specifically hybridize to a particularbacterial nucleic acid sequence. Hybridization of the PNA probe to anucleic acid sequence is indicative of the presence of the particulartype of bacteria of interest.

Direct sequence analysis can also be used to detect a bacterial nucleicacid of interest. A sample comprising DNA or RNA can be used, and PCR orother appropriate methods can be used to amplify all or a fragment ofthe nucleic acid, and/or its flanking sequences, if desired. Thebacterial nucleic acid, or a fragment thereof, is determined, usingstandard methods.

In another embodiment, arrays of oligonucleotide probes that arecomplementary to target microbial nucleic acid sequences can be used todetect and identify microbial nucleic acids. For example, in oneembodiment, an oligonucleotide array can be used. Oligonucleotide arraystypically comprise a plurality of different oligonucleotide probes thatare coupled to a surface of a substrate in different known locations.These oligonucleotide arrays, also known as “Genechips,” have beengenerally described in the art, for example, U.S. Pat. No. 5,143,854 andPCT patent publication Nos. WO 90/15070 and 92/10092. These arrays cangenerally be produced using mechanical synthesis methods or lightdirected synthesis methods which incorporate a combination ofphotolithographic methods and solid phase oligonucleotide synthesismethods. See Fodor et al., Science, 251:767-777 (1991), Pirrung et al.,U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070) andFodor et al., PCT Publication No. WO 92/10092 and U.S. Pat. No.5,424,186. Techniques for the synthesis of these arrays using mechanicalsynthesis methods are described in, e.g., U.S. Pat. No. 5,384,261.

After an oligonucleotide array is prepared, a nucleic acid of interestis hybridized with the array and scanned for particular bacterialnucleic acids. Hybridization and scanning are generally carried out bymethods described herein and also in, e.g., Published PCT ApplicationNos. WO 92/10092 and WO 95/11995, and U.S. Pat. No. 5,424,186, theentire teachings of which are incorporated by reference herein. Inbrief, a target bacterial nucleic acid sequence is amplified bywell-known amplification techniques, e.g., PCR. Typically, this involvesthe use of primer sequences that are complementary to the targetsequence. Amplified target, generally incorporating a label, is thenhybridized with the array under appropriate conditions. Upon completionof hybridization and washing of the array, the array is scanned todetermine the position on the array to which the target sequencehybridizes. The hybridization data obtained from the scan is typicallyin the form of fluorescence intensities as a function of location on thearray.

Other methods of nucleic acid analysis can be used to detect microbialnucleic acids of interest. Representative methods include direct manualsequencing (1988, Church and Gilbert, Proc. Natl. Acad. Sci. USA81:1991-1995; 1977, Sanger et al., Proc. Natl. Acad. Sci. 74:5463-5467;Beavis et al. U.S. Pat. No. 5,288,644); automated fluorescentsequencing; single-stranded conformation polymorphism assays (SSCP);clamped denaturing gel electrophoresis (CDGE); denaturing gradient gelelectrophoresis (DGGE) (1981, Sheffield et al., Proc. Natl. Acad. Sci.USA 86:232-236), mobility shift analysis (1989, Orita et al., Proc.Natl. Acad. Sci. USA 86:2766-2770; 1987, Rosenbaum and Reissner,Biophys. Chem. 265:1275; 1991, Keen et al., Trends Genet. 7:5);restriction enzyme analysis (1978, Flavell et al., Cell 15:25; 1981,Geever, et al., Proc. Natl. Acad. Sci. USA 78:5081); heteroduplexanalysis; chemical mismatch cleavage (CMC) (1985, Cotton et al., Proc.Natl. Acad. Sci. USA 85:4397-4401); RNase protection assays (1985,Myers, et al., Science 230:1242); use of polypeptides which recognizenucleotide mismatches, such as E. coli mutS protein (see, for example,U.S. Pat. No. 5,459,039); Luminex xMAP™ technology; high-throughputsequencing (HTS) (2011, Gundry and Vijg, Mutat Res,doi:10.1016/j.mrfmmm.2011.10.001); next-generation sequencing (NGS)(2009, Voelkerding et al., Clinical Chemistry 55:641-658; 2011, Su etal., Expert Rev Mol Diagn. 11:333-343; 2011, Ji and Myllykangas,Biotechnol Genet Eng Rev 27:135-158); ion semiconductor sequencing(2011, Rusk, Nature Methods doi:10.1038/nmeth.f.330; 2011, Rothberg etal., Nature 475:348-352) and/or allele-specific PCR, for example. Theseand other methods can be used to identify the presence of one or moremicrobial nucleic acids of interest, in a biological sample derived froma subject. In various embodiments of the invention, the methods ofassessing a biological sample for the presence or absence of aparticular nucleic acid sequence, as described herein, are used todetect, identify or quantify particular constituents of a subject'smicrobiota, and to aid in the diagnosis of an altered microbiotaassociated with an inflammatory disease or disorder in a subject in needthereof.

The probes and primers according to the invention can be labeleddirectly or indirectly with a radioactive or nonradioactive compound, bymethods well known to those skilled in the art, in order to obtain adetectable and/or quantifiable signal; the labeling of the primers or ofthe probes according to the invention is carried out with radioactiveelements or with nonradioactive molecules. Among the radioactiveisotopes used, mention may be made of ³²P, ³³P, ³⁵S or ³H. Thenonradioactive entities are selected from ligands such as biotin,avidin, streptavidin or digoxigenin, haptenes, dyes, and luminescentagents such as radioluminescent, chemiluminescent, bioluminescent,fluorescent or phosphorescent agents.

Nucleic acids can be obtained from the biological sample using knowntechniques. Nucleic acid herein refers to RNA, including mRNA, and DNA,including genomic DNA. The nucleic acid can be double-stranded orsingle-stranded (i.e., a sense or an antisense single strand) and can becomplementary to a nucleic acid encoding a polypeptide. The nucleic acidcontent may also be an RNA or DNA extraction performed on a fresh orfixed biological sample.

Routine methods also can be used to extract DNA from a biologicalsample, including, for example, phenol extraction. Alternatively,genomic DNA can be extracted with kits such as the QIAamp™. Tissue Kit(Qiagen, Chatsworth, Calif.), the Wizard™ Genomic DNA purification kit(Promega, Madison, Wis.), the Puregene DNA Isolation System (GentraSystems, Inc., Minneapolis, Minn.), and the A.S.A.P.™ Genomic DNAisolation kit (Boehringer Mannheim, Indianapolis, Ind.).

There are many methods known in the art for the detection of specificnucleic acid sequences and new methods are continually reported. A greatmajority of the known specific nucleic acid detection methods utilizenucleic acid probes in specific hybridization reactions. Preferably, thedetection of hybridization to the duplex form is a Southern blottechnique. In the Southern blot technique, a nucleic acid sample isseparated in an agarose gel based on size (molecular weight) and affixedto a membrane, denatured, and exposed to (admixed with) the labelednucleic acid probe under hybridizing conditions. If the labeled nucleicacid probe forms a hybrid with the nucleic acid on the blot, the labelis bound to the membrane.

In the Southern blot, the nucleic acid probe is preferably labeled witha tag. That tag can be a radioactive isotope, a fluorescent dye or theother well-known materials. Another type of process for the specificdetection of nucleic acids of exogenous organisms in a body sample knownin the art are the hybridization methods as exemplified by U.S. Pat.Nos. 6,159,693 and 6,270,974, and related patents. To briefly summarizeone of those methods, a nucleic acid probe of at least 10 nucleotides,preferably at least 15 nucleotides, more preferably at least 25nucleotides, having a sequence complementary to a desired region of thetarget nucleic acid of interest is hybridized in a sample, subjected todepolymerizing conditions, and the sample is treated with anATP/luciferase system, which will luminesce if the nucleic sequence ispresent. In quantitative Southern blotting, levels of the target nucleicacid can be determined.

A further process for the detection of hybridized nucleic acid takesadvantage of the polymerase chain reaction (PCR). The PCR process iswell known in the art (U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159). To briefly summarize PCR, nucleic acid primers,complementary to opposite strands of a nucleic acid amplification targetnucleic acid sequence, are permitted to anneal to the denatured sample.A DNA polymerase (typically heat stable) extends the DNA duplex from thehybridized primer. The process is repeated to amplify the nucleic acidtarget. If the nucleic acid primers do not hybridize to the sample, thenthere is no corresponding amplified PCR product. In this case, the PCRprimer acts as a hybridization probe.

In PCR, the nucleic acid probe can be labeled with a tag as discussedbefore. Most preferably the detection of the duplex is done using atleast one primer directed to the target nucleic acid. In yet anotherembodiment of PCR, the detection of the hybridized duplex compriseselectrophoretic gel separation followed by dye-based visualization.

DNA amplification procedures by PCR are well known and are described inU.S. Pat. No. 4,683,202. Briefly, the primers anneal to the targetnucleic acid at sites distinct from one another and in an oppositeorientation. A primer annealed to the target sequence is extended by theenzymatic action of a heat stable DNA polymerase. The extension productis then denatured from the target sequence by heating, and the processis repeated. Successive cycling of this procedure on both DNA strandsprovides exponential amplification of the region flanked by the primers.

Amplification is then performed using a PCR-type technique, that is tosay the PCR technique or any other related technique. Two primers,complementary to the target nucleic acid sequence are then added to thenucleic acid content along with a polymerase, and the polymeraseamplifies the DNA region between the primers.

The expression “specifically hybridizing in stringent conditions” refersto a hybridizing step in the process of the invention where theoligonucleotide sequences selected as probes or primers are of adequatelength and sufficiently unambiguous so as to minimize the amount ofnon-specific binding that may occur during the amplification. Theoligonucleotide probes or primers herein described may be prepared byany suitable methods such as chemical synthesis methods.

Hybridization is typically accomplished by annealing the oligonucleotideprobe or primer to the DNA under conditions of stringency that preventnon-specific binding but permit binding of this DNA which has asignificant level of homology with the probe or primer.

Among the conditions of stringency is the melting temperature (Tm) forthe amplification step using the set of primers, which is in the rangeof about 55° C. to about 70° C. Preferably, the Tm for the amplificationstep is in the range of about 59° C. to about 72° C. Most preferably,the Tm for the amplification step is about 60° C.

Typical hybridization and washing stringency conditions depend in parton the size (i.e., number of nucleotides in length) of the DNA or theoligonucleotide probe, the base composition and monovalent and divalentcation concentrations (Ausubel et al., 1997, eds Current Protocols inMolecular Biology).

In a preferred embodiment, the process for determining the quantitativeand qualitative profile according to the present invention ischaracterized in that the amplifications are real-time amplificationsperformed using a labeled probe, preferably a labeled hydrolysis-probe,capable of specifically hybridizing in stringent conditions with asegment of a nucleic acid sequence, or polymorphic nucleic acidsequence. The labeled probe is capable of emitting a detectable signalevery time each amplification cycle occurs.

The real-time amplification, such as real-time PCR, is well known in theart, and the various known techniques will be employed in the best wayfor the implementation of the present process. These techniques areperformed using various categories of probes, such as hydrolysis probes,hybridization adjacent probes, or molecular beacons. The techniquesemploying hydrolysis probes or molecular beacons are based on the use ofa fluorescence quencher/reporter system, and the hybridization adjacentprobes are based on the use of fluorescence acceptor/donor molecules.

Hydrolysis probes with a fluorescence quencher/reporter system areavailable in the market, and are for example commercialized by theApplied Biosystems group (USA). Many fluorescent dyes may be employed,such as FAM dyes (6-carboxy-fluorescein), or any other dyephosphoramidite reagents.

Among the stringent conditions applied for any one of thehydrolysis-probes of the present invention is the Tm, which is in therange of about 65° C. to 75° C. Preferably, the Tm for any one of thehydrolysis-probes of the present invention is in the range of about 67°C. to about 70° C. Most preferably, the Tm applied for any one of thehydrolysis-probes of the present invention is about 67° C.

In another preferred embodiment, the process for determining thequantitative and qualitative profile according to the present inventionis characterized in that the amplification products can be elongated,wherein the elongation products are separated relative to their length.The signal obtained for the elongation products is measured, and thequantitative and qualitative profile of the labeling intensity relativeto the elongation product length is established.

The elongation step, also called a run-off reaction, allows one todetermine the length of the amplification product. The length can bedetermined using conventional techniques, for example, using gels suchas polyacrylamide gels for the separation, DNA sequencers, and adaptedsoftware. Because some mutations display length heterogeneity, somemutations can be determined by a change in length of elongationproducts.

In one aspect, the invention includes a primer that is complementary toa target bacterial nucleic acid, and more particularly the primerincludes 12 or more contiguous nucleotides substantially complementaryto the sequence flanking the nucleic acid sequence of interest.Preferably, a primer featured in the invention includes a nucleotidesequence sufficiently complementary to hybridize to a nucleic acidsequence of about 12 to 25 nucleotides. More preferably, the primerdiffers by no more than 1, 2, or 3 nucleotides from the target flankingnucleotide sequence. In another aspect, the length of the primer canvary in length, preferably about 15 to 28 nucleotides in length (e.g.,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides inlength).

The present invention also pertains to kits useful in the methods of theinvention. Such kits comprise components useful in any of the methodsdescribed herein, including for example, hybridization probes or primers(e.g., labeled probes or primers), reagents for detection of labeledmolecules, means for amplification of nucleic acids, means for analyzinga nucleic acid sequence, and instructional materials. For example, inone embodiment, the kit comprises components useful for analysis of abacterial nucleic acid of interest present in a biological sampleobtained from a subject. In a preferred embodiment of the invention, thekit comprises components for detecting one or more of the bacterialnucleic acids of interest present in a biological sample derived from asubject.

Methods of Treatment

In some embodiments, the invention relates to methods of modifying analtered microbiota having secretory antibody-coated constituents in asubject in need thereof. In some embodiments, the invention providescompositions and methods for supplementing constituents of an alteredmicrobiota that are under-represented in the altered microbiota, ascompared with a normal microbiota, to restore the subject's microbiotato a normal microbiota. In other embodiments, the invention providescompositions and methods for diminishing constituents of an alteredmicrobiota that are over-represented in the altered microbiota ascompared with a normal microbiota, such as over-represented secretoryantibody-coated constituents, to restore the subject's microbiota to anormal microbiota. As used throughout herein, constituents of an alteredmicrobiota that are over-represented in the altered microbiota ascompared with a normal microbiota, include constituents that areuniquely present in the altered microbiota as compared with a normalmicrobiota. In further embodiments, the invention provides compositionsand methods for both supplementing constituents of an altered microbiotathat are under-represented in the altered microbiota, as well asdiminishing constituents of an altered microbiota that areover-represented in the altered microbiota, as compared with a normalmicrobiota, to restore the subject's microbiota to a normal microbiota.The microbiota of the subject can be any microbiota present on anymucosal surface of subject where antibody is secreted, including thegastrointestinal tract, the respiratory tract, genitourinary tract andmammary gland.

In conjunction with the diagnostic methods, the present invention alsoprovides therapeutic methods for treating an inflammatory disease ordisorder associated with an altered microbiota including secretoryantibody-coated microbes, by modifying the microbiota to that observedin a healthy subject. In some embodiments, the methods supplement thenumbers of the types of microbes that are under-represented in thealtered microbiota. In other embodiments, the methods diminish thenumbers of the types of microbes, including secretory antibody-coatedmicrobes that are overrepresented in the altered microbiota. In afurther embodiment, the methods both supplement the numbers of the typesof bacteria that are under-represented in the altered microbiota, anddiminish the numbers of the types of bacteria that are overrepresentedin the altered microbiota. In various embodiments, the inflammatorydiseases and disorders treatable by the methods of the inventioninclude, but are not limited to: inflammatory bowel disease, celiacdisease, colitis, intestinal hyperplasia, metabolic syndrome, obesity,rheumatoid arthritis, liver disease, hepatic steatosis, fatty liverdisease, non-alcoholic fatty liver disease (NAFLD), or non-alcoholicsteatohepatitis (NASH).

In some embodiments, modification of the altered microbiota is achievedby administering to a subject in need thereof a therapeuticallyeffective amount of a probiotic composition comprising an effectiveamount of at least one type of bacteria, or a combinations of severaltypes of bacteria, wherein the administered bacteria supplements thenumber of the types of bacteria which are under-represented in thealtered microbiota, as compared with a normal microbiota.

Bacteria administered according to the methods of the present inventioncan comprise live bacteria. One or several different types of bacteriacan be administered concurrently or sequentially. Such bacteria can beobtained from any source, including being isolated from a microbiota andgrown in culture using known techniques.

In certain embodiments, the administered bacteria used in the methods ofthe invention further comprise a buffering agent. Examples of usefulbuffering agents include sodium bicarbonate, milk, yogurt, infantformula, and other dairy products.

Administration of a bacterium can be accomplished by any method suitablefor introducing the organisms into the desired location. The bacteriacan be mixed with a carrier and (for easier delivery to the digestivetract) applied to a liquid or to food. The carrier material should benon-toxic to the bacteria as wells as the subject. Preferably, thecarrier contains an ingredient that promotes viability of the bacteriaduring storage. The formulation can include added ingredients to improvepalatability, improve shelf-life, impart nutritional benefits, and thelike.

The dosage of the administered bacteria will vary widely, depending uponthe nature of the inflammatory disease or disorder, the character ofsubject's altered microbiota, the subject's medical history, thefrequency of administration, the manner of administration, the clearanceof the agent from the host, and the like. The initial dose may belarger, followed by smaller maintenance doses. The dose may beadministered as infrequently as weekly or biweekly, or fractionated intosmaller doses and administered daily, semi-weekly, etc., to maintain aneffective dosage level. It is contemplated that a variety of doses willbe effective to achieve colonization of the gastrointestinal tract withthe desired bacteria. In some embodiments, the dose ranges from about10⁶ to about 10¹⁰ CFU per administration. In other embodiments, the doseranges from about 10⁴ to about 10⁶ CFU per administration.

In certain embodiments, the present invention relates to a method formodifying an altered microbiota comprising administering to a subject inneed of such treatment, an effective amount of at least one gastric,esophageal, or intestinal bacterium, or combinations thereof. In apreferred embodiment, the bacteria are administered orally.Alternatively, bacteria can be administered rectally or by enema.

The organisms contemplated for administration to modify the alteredmicrobiota include any of the bacteria identified herein asunder-represented in an altered microbiota. One of the organismscontemplated for administration to modify the altered microbiota is atleast one Lactobacillus spp. In certain embodiments, the bacteriaadministered in the therapeutic methods of the invention compriseadministration of a combination of organisms.

While it is possible to administer a bacteria for therapy as is, it maybe preferable to administer it in a pharmaceutical formulation, e.g., inadmixture with a suitable pharmaceutical excipient, diluent or carrierselected with regard to the intended route of administration andstandard pharmaceutical practice. The excipient, diluent and/or carriermust be “acceptable” in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipientthereof. Acceptable excipients, diluents, and carriers for therapeuticuse are well known in the pharmaceutical art, and are described, forexample, in Remington: The Science and Practice of Pharmacy. LippincottWilliams & Wilkins (A. R. Gennaro edit. 2005). The choice ofpharmaceutical excipient, diluent, and carrier can be selected withregard to the intended route of administration and standardpharmaceutical practice.

Although there are no physical limitations to delivery of theformulations of the present invention, oral delivery is preferred fordelivery to the digestive tract because of its ease and convenience, andbecause oral formulations readily accommodate additional mixtures, suchas milk, yogurt, and infant formula. For delivery to colon, bacteria canbe also administered rectally or by enema.

In other embodiments, modification of the altered microbiota havingover-represented secretory antibody-coated constituents is achieved byadministering to a subject in need thereof a therapeutically effectiveamount of a vaccine to induce an immune response against theover-represented constituent, wherein the administered vaccine andensuing immune response diminishes the number of at least one type ofsecretory antibody-coated bacteria that is over-represented in thealtered microbiota, as compared with a normal microbiota. In variousembodiments, the at least one type of bacteria that is diminished usingthe methods of the invention includes a Segmented Filamentous Bacteria(SFB) or Helicobacter flexispira, or a bacteria from at least one familyselected from the group consisting of Lactobacillus, Helicobacter,S24-7, Erysipelotrichaceae and Prevotellaceae. In some embodiments, thebacteria from the family Prevotellaceae is a bacteria from the genera ofParaprevotella or Prevotella. In other embodiments, the secretoryantibody-coated constituent of the subject's microbiota associated withthe development or progression of an inflammatory disease or disorder inthe subject is at least one selected from Eubacterium, Eubacteriumbiforme, Eubacterium dolichum, Ruminococcus gnavus, Acidaminococcus,Actinomyces, Allobaculum, Anaerostipes, Bacteroides, Bacteroidesfragilis, Bacteroides Other, Bifidobacterium, Bifidobacteriumadolescentis, Bifidobacterium Other, Blautia, Blautia obeum, BlautiaOther, Blautia producta, Bulleidia Other, Clostridium, Clostridiumperfringens, Collinsella aerofaciens, Coprococcus, Coprococcus catus,Dialister, Eggerthella lenta, Erysipelotrichaceae, Faecalibacteriumprausnitzii, Haemophilus parainfluenzae, Lachnospiraceae,Lachnospiraceae other, Lactobacillus, Lactobacillus mucosae,Lactobacillus Other, Lactobacillus reuteri, Lactobacillus zeae,Oscillospira, Pediococcus Other, Rikenellaceae, Roseburia, Roseburiafaecis, Ruminococcaceae, Ruminococcus, Ruminococcus bromii, SMB53,Streptococcus, Streptococcus luteciae, Streptococcus Other, Sutterella,Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UCRuminococcaceae, Veillonella, Veillonella dispar, and Weissella.

In other embodiments, modification of the altered microbiota havingover-represented secretory antibody-coated constituents is achieved byadministering to a subject in need thereof a therapeutically effectiveamount of a passive immunotherapy or passive vaccine, such as by theadministration of immunoglobulin (e.g., IgA) against theover-represented constituent, wherein the administered passive vaccineand ensuing immune response diminishes the number of at least one typeof secretory antibody-coated bacteria that is over-represented in thealtered microbiota, as compared with a normal microbiota. In someembodiments, the immunoglobulin is administered orally. Alternatively,the immunoglobulin can be administered rectally or by enema. In variousembodiments, the at least one type of bacteria that is diminished usingthe methods of the invention includes a Segmented Filamentous Bacteria(SFB) or Helicobacter flexispira, or a bacteria from at least one familyselected from the group consisting of Lactobacillus, Helicobacter,S24-7, Erysipelotrichaceae and Prevotellaceae. In some embodiments, thebacteria from the family Prevotellaceae is a bacteria from the genera ofParaprevotella or Prevotella. In other embodiments, the secretoryantibody-coated constituent of the subject's microbiota associated withthe development or progression of an inflammatory disease or disorder inthe subject is at least one selected from Eubacterium, Eubacteriumbiforme, Eubacterium dolichum, Ruminococcus gnavus, Acidaminococcus,Actinomyces, Allobaculum, Anaerostipes, Bacteroides, Bacteroidesfragilis, Bacteroides Other, Bifidobacterium, Bifidobacteriumadolescentis, Bifidobacterium Other, Blautia, Blautia obeum, BlautiaOther, Blautia producta, Bulleidia Other, Clostridium, Clostridiumperfringens, Collinsella aerofaciens, Coprococcus, Coprococcus catus,Dialister, Eggerthella lenta, Erysipelotrichaceae, Faecalibacteriumprausnitzii, Haemophilus parainfluenzae, Lachnospiraceae,Lachnospiraceae other, Lactobacillus, Lactobacillus mucosae,Lactobacillus Other, Lactobacillus reuteri, Lactobacillus zeae,Oscillospira, Pediococcus Other, Rikenellaceae, Roseburia, Roseburiafaecis, Ruminococcaceae, Ruminococcus, Ruminococcus bromii, SMB53,Streptococcus, Streptococcus luteciae, Streptococcus Other, Sutterella,Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UCRuminococcaceae, Veillonella, Veillonella dispar, and Weissella.

In other embodiments, modification of the altered microbiota havingover-represented secretory antibody-coated constituents is achieved byadministering to a subject in need thereof a therapeutically effectiveamount of antibiotic composition comprising an effective amount of atleast one antibiotic, or a combinations of several types of antibiotics,wherein the administered antibiotic diminishes the number of at leastone type of secretory antibody-coated bacteria that is over-representedin the altered microbiota, as compared with a normal microbiota. Invarious embodiments, the at least one type of bacteria that isdiminished using the methods of the invention includes a SegmentedFilamentous Bacteria (SFB) or Helicobacter flexispira, or a bacteriafrom at least one family selected from the group consisting ofLactobacillus, Helicobacter, S24-7, Erysipelotrichaceae andPrevotellaceae. In some embodiments, the bacteria from the familyPrevotellaceae is a bacteria from the genera of Paraprevotella orPrevotella. In other embodiments, the secretory antibody-coatedconstituent of the subject's microbiota associated with the developmentor progression of an inflammatory disease or disorder in the subject isat least one selected from Eubacterium, Eubacterium biforme, Eubacteriumdolichum, Ruminococcus gnavus, Acidaminococcus, Actinomyces,Allobaculum, Anaerostipes, Bacteroides, Bacteroides fragilis,Bacteroides Other, Bifidobacterium, Bifidobacterium adolescentis,Bifidobacterium Other, Blautia, Blautia obeum, Blautia Other, Blautiaproducta, Bulleidia Other, Clostridium, Clostridium perfringens,Collinsella aerofaciens, Coprococcus, Coprococcus catus, Dialister,Eggerthella lenta, Erysipelotrichaceae, Faecalibacterium prausnitzii,Haemophilus parainfluenzae, Lachnospiraceae, Lachnospiraceae other,Lactobacillus, Lactobacillus mucosae, Lactobacillus Other, Lactobacillusreuteri, Lactobacillus zeae, Oscillospira, Pediococcus Other,Rikenellaceae, Roseburia, Roseburia faecis, Ruminococcaceae,Ruminococcus, Ruminococcus bromii, SMB53, Streptococcus, Streptococcusluteciae, Streptococcus Other, Sutterella, Turicibacter, UCClostridiales, UC Erysipelotrichaceae, UC Ruminococcaceae, Veillonella,Veillonella dispar, and Weissella.

The type and dosage of the administered antibiotic will vary widely,depending upon the nature of the inflammatory disease or disorder, thecharacter of subject's altered microbiota, the subject's medicalhistory, the frequency of administration, the manner of administration,and the like. The initial dose may be larger, followed by smallermaintenance doses. The dose may be administered as infrequently asweekly or biweekly, or fractionated into smaller doses and administereddaily, semi-weekly, etc., to maintain an effective dosage level. Invarious embodiments, the administered antibiotic is at least one oflipopeptide, fluoroquinolone, ketolide, cephalosporin, amikacin,gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin,tobramycin, cefacetrile, cefadroxil, cefalexin, cefaloglycin,cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur,cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor,cefamandole, cefmetazole, cefonicid, cefotetan, cefoxitin, cefprozil,cefuroxime, cefuzonam, cefcapene, cefdaloxime, cefdinir, cefditoren,cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefpimizole,cefpodoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime,ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepimecefluprenam, cefoselis, cefozopran, cefpirome, cefquinome,cefaclomezine, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone,cefetrizole, cefivitril, cefmatilen, cefmepidium, cefovecin, cefoxazole,cefrotil, cefsumide, ceftaroline, ceftioxide, cefuracetime, imipenem,primaxin, doripenem, meropenem, ertapenem, flumequine, nalidixic acid,oxolinic acid, piromidic acid pipemidic acid, rosoxacin, ciprofloxacin,enoxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin,pefloxacin, rufloxacin, balofloxacin, gatifloxacin, grepafloxacin,levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin,tosufloxacin, clinafloxacin, gemifloxacin, sitafloxacin, trovafloxacin,prulifloxacin, azithromycin, erythromycin, clarithromycin,dirithromycin, roxithromycin, telithromycin, amoxicillin, ampicillin,bacampicillin, carbenicillin, cloxacillin, dicloxacillin,flucloxacillin, mezlocillin, nafcillin, oxacillin, penicillin g,penicillin v, piperacillin, pivampicillin, pivmecillinam, ticarcillin,sulfamethizole, sulfamethoxazole, sulfisoxazole,trimethoprim-sulfamethoxazole, demeclocycline, doxycycline, minocycline,oxytetracycline, tetracycline, linezolid, clindamycin, metronidazole,vancomycin, vancocin, mycobutin, rifampin, nitrofurantoin,chloramphenicol, or derivatives thereof.

In a further embodiment, modification of the altered microbiota isachieved by both administering at least one type of bacteria tosupplement the numbers of at least one type of bacteria that isunder-represented in the altered microbiota, and administering at leastone antibiotic to diminish the numbers of at least one type of bacteriathat is over-represented in the altered microbiota.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: IgA Coating of the Intestinal Microbiota in Health andDisease

Immunoglobulin A represents one of the major mechanisms by which theadaptive immune system controls commensal bacteria in thegastrointestinal tract. However, little is known about the specificityof the intestinal IgA response. To better understand interactionsbetween the immune system and the microbiota and to identify intestinalbacteria that preferentially stimulate immune responses, IgA coating ofthe intestinal microbiota was examined by separating IgA-coated andnon-coated bacteria and performing 16S rRNA sequencing. While it wasfound that each individual displays a unique set of IgA coated bacterialspecies, IgA coating of select bacterial taxa was conserved within thehealthy human population. Notably, inflammatory bowel disease (IBD)patients and mice with a colitogenic dysbiosis displayed dramaticallyaltered patterns of IgA coating, suggesting dysregulation ofhost-microbiota homeostasis, and showed high coating of bacterialspecies that were uniquely present in dysbiosis or in patients with IBD.Given the importance of IgA in maintenance of host-microbiota symbiosis,highly IgA-coated taxa appear to play novel and critical roles inshaping the immune response and host physiology in health and disease.

Described herein is a novel technique named IgA-SEQ that combines flowcytometry-based bacterial cell sorting and 16S ribosomal RNA-basedmetagenomic analyses to sort and identify IgA-coated bacterial speciesfrom the intestinal microbiota. Because many disease-causing members ofthe microbiota may cause disease because they trigger the immuneresponse, including IgA production and secretion, IgA-SEQ can accuratelyidentify bacteria that are responsible for driving disease developmentin populations or in a particular individual. Therefore, in the methodsdescribed herein, the immune response is utilized to identify putativedisease-causing bacteria by determining which bacterial species aretargeted by an individual host's immune system. The methods describedherein exploit the host's immune response to the microbiota to identifydisease-causing bacteria in the intestine in an individualized manner.Although the methods described herein are useful at the individuallevel, patterns and trends identified in multiple individuals can beused to identify characteristics of populations of individuals. IgA-SEQwas shown to accurately identify disease-causing members of themicrobiota in mice with a microbiota that exacerbates colitis. Thistechnique is also useful to identify disease-causing bacteria inpatients with a variety of other inflammatory diseases and disorders,including, by way of non-limiting example, Crohn's disease.

The materials and methods employed in these experiments are nowdescribed.

Animals

ASC-deficient mice were bred and maintained at the Yale UniversitySchool of Medicine and all treatments were in accordance with YaleAnimal Care and Use Committee guidelines. Wild type mice were from theNational Cancer Institute (NCI; Charles River).

Inflammasome-Mediated Intestinal Dysbiosis

Intestinal dysbiosis was induced by co-housing wild type C57Bl/6 micefrom NCI with ASC-deficient mice at a 1 to 1 ratio for at least 6 weeks.

DSS Colitis

SPF and SPF^(dysbiosis) mice were treated with 2% Dextran Sodium Sulfate(MP Biomedicals) in the drinking water ad libitum for 7 days to inducecolitis. Weight was measured each day for 14 days.

ELISA Pre-sort, IgA+ and IgA− fractions (after MACS sorting) were probedfor IgA by ELISA (Coating: MP Biomedicals 55478, Detection: SigmaB2766).

Fecal IgA Flow Cytometry

Fecal pellets from mice or ˜100 mg of human fecal material werecollected in Fast Prep Lysing Matrix D tubes containing ceramic beads(MP Biomedicals) and incubated in 1 mL Phosphate Buffered Saline (PBS)per 100 mg fecal material on ice for 1 hour. Fecal pellets werehomogenized by bead beating for 5 seconds (Minibeadbeater; Biospec) andcentrifuged (50×g, 15 min, 4° C.) to remove large particles. Fecalbacteria in the supernatants were removed (100 μl/sample), washed with 1mL PBS containing 1% (w/v) Bovine Serum Albumin (BSA, AmericanBioanalytical; staining buffer) and centrifuged for 5 min (8,000×g, 4°C.) before resuspension in 1 mL staining buffer. A small sample of thisbacterial suspension (20 μl) was saved as the Pre-sort sample for 16Ssequencing analysis. After an additional wash, bacterial pellets wereresuspended in 100 μl blocking buffer (staining buffer containing 20%Normal Rat Serum for mouse samples or 20% Normal Mouse Serum for humansamples, both from Jackson ImmunoResearch), incubated for 20 min on ice,and then stained with 100 μl staining buffer containing PE-conjugatedAnti-Mouse IgA (1:12.5; eBioscience clone mA-6E1) or PE-conjugatedAnti-Human IgA (1:10; Miltenyi Biotec clone IS11-8E10) for 30 minutes onice. Samples were then washed 3 times with 1 mL staining buffer beforeflow cytometric analysis or cell separation.

Sorting of IgA+ and IgA− Bacteria from Feces

Anti-IgA stained fecal bacteria were incubated in 1 ml staining buffercontaining 50 μl Anti-PE Magnetic Activated Cell Sorting (MACS) beads(Miltenyi Biotec) (15 min at 4° C.), washed twice with 1 ml StainingBuffer (10,000×g, 5 min, 4° C.), and then sorted by MACS (Possel_sprogram on an AutoMACS pro; Miltenyi). After MACS separation, 50 μl ofthe negative fraction was collected for 16S sequencing analysis(IgA-negative fraction). The positive fraction was then further purifiedvia Fluorescence Activated Cell Sorting (FACSAria; BD Biosciences). Foreach sample, 2 million IgA-positive bacteria were collected, pelleted(10,000×g, 5 min, 4° C.), and frozen along with the Pre-sort andIgA-negative samples at −80° C. until 16S sequencing analysis.

Bacterial DNA Purification and 16S V4 PCR Amplification

All bacterial samples were suspended in 400 μl staining buffer beforeadding 250 μl 0.1 mm zirconia/silica beads (Biospec), 300 μl Lysisbuffer (200 mM NaCl, 200 mM Tris, 20 mM EDTA, pH 8), 200 μl 20% SDS and500 μl phenol:chloroform:isoamylalcohol (25:24:1, pH 7.9; Sigma).Samples were chilled on ice for 4 min and homogenized by beat beating (2min bead beating, 2 min on ice, 2 min bead beating). Aftercentrifugation (8000 rpm, 4° C.), the aqueous phase was transferred to aPhase Lock Gel tube (Light; 5 PRIME), an equal volume ofphenol:chloroform:isoamylalcohol was added, samples were mixed byinversion and centrifuged for 3 min (13,200 rpm, room temperature). TheDNA was then precipitated by adding 1/10 volume of 3M NaOAc (pH5.5) and1 volume Isopropanol to the aqueous phase, followed by incubation at−20° C. overnight. Precipitated DNA was pelleted (20 min, 13,200 rpm, 4°C.), washed with 500 μl 100% EtOH (3 min, 13,200 rpm, 4° C.), dried(miVac GeneVac 15 min, no heat, Auto Run setting), resuspended in 100 μlof TE buffer (pH 7) and incubated at 50° C. for 30 min. The DNA was thentreated with 35 U/ml RNase A (Qiagen) before purification (QIAquick PCRpurification; Qiagen) and elution in 40 μl Elution Buffer. The V4 regionof 16S ribosomal RNA was PCR amplified (28 cycles) in triplicate frompurified DNA (10 μl per reaction) using Phusion polymerase (New EnglandBioscience) and the primer pair F515/R806. After amplification, PCRtriplicates were pooled, purified using a MinElute kit (Qiagen), andresuspended in 20 μl H₂O. PCR products were then quantified viafluorimetry using Picogreen (Invitrogen) and pooled at a finalconcentration of 10 nM before sequencing using a miSeq sequencer(Illumina, 2×250 bp paired-end reads, up to 200 samples per sequencingrun).

16S and Statistical Analysis

Paired end reads were assembled with a novel pipeline that usesPANDA-seq (Masella et al., 2012, BMC bioinformatics 13:31) and assignsconsensus Q scores to the assembled reads. Microbial diversity wasanalyzed using the Quantitative Insights Into Microbial Ecology (QIIMEversion 1.7) analysis suite. Reads were demultiplexed and qualityfiltered (Q-score cutoff 30), then clustered into 97% identityOperational Taxonomic Units (OTUs) using the open-reference OTU pickingworkflow in QIIME and the Greengenes reference OTU database. Taxonomywas assigned to representative OTUs using the Ribosomal Database Projectclassifier (RDP) and the May 2013 Greengenes taxonomy (Caporaso et al.,2010, Nature methods 7:335; Wang et al., 2007, Applied and environmentalmicrobiology 73:5261; Lozupone et al., 2005, Applied and environmentalmicrobiology 71:8228). OTUs of less than 0.01% relative abundance andcontaminating OTUs that were also found after sequencing of 16Samplicons from PCR samples without template DNA, were filtered from OTUtables. Filtered OTU tables were rarefied to a depth of 5000 sequencesper sample for all further analyses.

All microbial ecology analyses (beta diversity, PCoA, PERMANOVA/adonis)were performed using QIIME and the Vegan package for R (version 2.1-21).Linear Discriminant Analysis Effect Size (LEfSe) analyses were performedusing the LEfSe Galaxy module (huttenhower.sph.harvard.edu/galaxy).Wilcoxon rank-sum tests were performed using R. Taxa that wereundetectable in both the IgA+ and IgA− fractions in a given sample wereconsidered not present and were assigned as missing-values for Wilcoxonrank-sum tests. As LEfSe cannot handle missing values, thesemissing-values were replaced with zeros for all LEfSe analyses. To allowfor the calculation of ICI scores for taxa that were undetectable in theIgA− fraction but detected in the IgA+ fraction, and which are thereforehighly-coated, zeroes in the negative fraction were replaced with arelative abundance of 0.0002, which is the limit of detection (1sequence in 5000).

Human Fecal Samples

The human study protocol was approved by the Institutional Review Board(Protocol No.10-1047) of the Icahn Medical School at Mount Sinai, N.Y.The healthy subjects were recruited through the Mount Sinai Biobank oran advertisement. Fresh fecal samples were collected at home, stored at−20° C. in an insulated foam shipper, mailed to Mount Sinai overnightand then stored at −80° C. for further analysis. A short questionnairewas also administrated to collect participants' health information.

The results of the experiments are now described.

IgA-SEQ Analysis

The studies described herein examine IgA coating of the intestinalmicrobiota in a comprehensive and unbiased manner in order tocharacterize patterns of IgA coating and determine if there are membersof the microbiota that are preferentially recognized and targeted by themucosal immune system. Therefore, an approach was devised combiningantibody-based bacterial cell sorting and 16S ribosomal RNA genesequencing to specifically isolate and identify IgA coated bacteria fromfecal material (IgA-SEQ, FIG. 1A). First, staining of fecal bacteriafrom Specific Pathogen Free (SPF) mice for IgA confirmed that only afraction of intestinal bacteria are measurably IgA coated, as determinedby flow cytometry (FIGS. 4A and 4B); importantly, intestinal bacteriafrom recombination activating gene 2 (Rag2)-deficient mice, which cannotproduce antibodies, showed negligible staining for IgA. IgA coated(IgA+) and non-coated (IgA−) bacteria were subsequently isolated using acombination of magnetic activated cell sorting (MACS) and fluorescenceactivated cell sorting (FACS) and confirmed the specificity and efficacyof the sorting by reanalyzing sorted fractions via flow cytometry (FIG.1B; and FIG. 5A) and ELISA (FIG. 5B). Principal Coordinates Analysis(PCoA) and PERMANOVA analysis of weighted UniFrac distances of 16S rRNAsequences of total, IgA+ and IgA− fecal bacteria revealed that, insteadof comprising a random sampling of all intestinal bacteria, IgA coatedbacteria represent a phylogenetically distinct sub-community of themicrobiota, as compared to total fecal bacteria or non-coated bacteria(P<0.05) (FIG. 1C and FIG. 5C). In contrast, total fecal bacteria andnon-coated bacteria were not significantly different. Importantly,sorting by itself did not appreciably alter the observed microbialcomposition since PCoA and PERMANOVA analysis of weighted UniFracdistances showed no significant differences between mock sorted samplesand the total microbiota (FIG. 1C and FIG. 5C). These data demonstratethat IgA coating of the microbiota is selective across microbial taxa,and show that IgA-coated bacteria represent a taxonomically distinctsubset of intestinal bacteria in SPF mice.

To identify which specific bacterial taxa were highly coated with IgA,relative abundances of bacterial genera were examined in total, IgAcoated bacteria and non-coated bacteria isolated from the feces of SPFmice (FIG. 2A; and FIG. 6). To quantify and compare relative levels ofIgA coating between taxa, an IgA Coating Index (ICI) was calculated foreach individual bacterial taxon as follows:ICI=IgA+^(relative abundance)/IgA-^(relative abundance). Taxonomicabundance in the IgA+ and IgA− fractions were then compared using boththe Wilcoxon rank-sum test and Linear Discriminant Analysis Effect Size(LEfSe; Segata et al., 2011, Genome biology 12:R60), which combines theKruskal-Wallis sum-rank test, Wilcoxon rank-sum test and LinearDiscriminant Analysis, to determine which taxa were enriched in eitherthe IgA+ or IgA− fractions (FIG. 6B). Taxa that were significantlyhigher in the IgA+ fraction were classified as highly coated, whereastaxa that were significantly higher in the IgA− fraction were classifiedas low or non-coated. These analyses revealed that four genera werehighly coated with IgA in SPF mice (P<0.05, LEfSe and Wilcoxonrank-sum): an unclassified genus of the family S24-7 from the orderBacteroidales, Lactobacillus, Segmented Filamentous Bacteria (SFB), andunclassified Erysipelotrichaceae. In addition, 22 taxa showed low or nocoating with IgA (P<0.05, LEfSe and Wilcoxon rank-sum), while theremaining taxa were neither enriched nor depleted by IgA-basedseparation. These data demonstrate that only a small number of bacterialtaxa are highly coated with IgA in mice with a complex microbiota.

The inflammasome is a critical component of the innate immune systemthat orchestrates the activation of Caspase-1 and release of theinflammatory cytokines IL-1β and IL-18 in response to infection (Strowiget al. 2012, Nature 481:278). It was recently shown that mice lackingcomponents of the inflammasome, such as the signaling adaptorapoptosis-associated speck-like protein containing a CARD (ASC), harbora dysbiotic intestinal microbiota that can be transmitted to wild typeSPF mice through co-housing (Elinav et al., 2011, Cell 145:745). Thisinflammasome-mediated dysbiosis is characterized by the expansion ofPrevotellaceae species, which leads to an increase in the severity ofchemically-induced colitis. To identify bacteria that are highly coatedwith IgA in the context of a colitogenic microbiota, IgA-SEQ wasperformed on SPF mice that had acquired inflammasome-mediated dysbiosisthrough co-housing with Asc^(−/−) mice (SPF^(dysbiosis)). As previouslyreported, co-housing altered the composition of the intestinalmicrobiota and increased susceptibility to colitis (FIG. 7A to 7C). Flowcytometric analysis of IgA coating of the intestinal microbiota ofSPF^(dysbiosis) mice revealed an increase in the total percentage ofintestinal bacteria coated with IgA as compared to SPF mice, suggestingthat acquisition of dysbiosis may alter the pattern or extent of IgAcoating (FIG. 7D). This was confirmed by PCoA and PERMANOVA analysis ofweighted UniFrac distances of total, IgA+ and IgA− fractions, whichshowed that IgA coated bacteria in SPF^(dysbiosis) mice arephylogenetically distinct from non-coated bacteria and from IgA coatedbacteria in healthy SPF mice (FIG. 8A; P<0.05, PERMANOVA).

While 19 taxa in SPF^(dysbiosis) mice showed significant expansion as aresult of co-housing, only two of these taxa were highly coated with IgA(FIG. 2B, and FIGS. 8B and 8C; P<0.05 LEfSe and Wilcoxon rank-sum); themost abundant IgA coated taxon was an unclassified genus from thePrevotellaceae family, which is the defining taxon ininflammasome-mediated dysbiosis (Elinav et al., 2011, Cell 145:745).Helicobacter sp. flexispira, which was acquired during co-housing, wasalso significantly enriched in the IgA coated fraction inSPF^(dysbiosis) mice. Finally, as in SPF mice, Lactobacillus and SFBremain significantly enriched in the IgA coated fraction in mice withintestinal dysbiosis.

Taken together, only six bacterial taxa were found to be highly coatedwith IgA in SPF or SPF^(dysbiosis) mice: an unclassified genus of S24-7from the order Bacteroidales, Lactobacillus, SFB, unclassifiedErysipelotrichaceae, Helicobacter sp. flexispira, and an unclassifiedPrevotellaceae. This implies that the immune responses that weredetected using IgA-SEQ are specific to bacterial antigens that areunique to particular members of the microbiota, rather than sharedacross bacterial taxa. The induction of such responses requires intimateinteraction between the mucosal immune system and target bacteria.Indeed, IgA has been specifically implicated in controlling members ofthe microbiota that penetrate the mucus layer and attach to theintestinal epithelium, including SFB (Suzuki et al., 2004, 101:1981);such bacteria are natural targets for the mucosal immune response astheir location close to the epithelium signals a potential threat andenables efficient sampling by cells of the intestinal immune system. Toexplore the possibility that IgA coated bacteria are mucus-associated,mucus-associated and luminal bacteria were isolated from the smallintestine, cecum and colon of SPF^(dysbiosis) mice and the distributionof IgA coated bacteria in these fractions was analyzed (FIG. 9). Smallintestinal, cecal and large intestinal bacteria (e.g., SFB,Prevotellaceae, and Helicobacter sp. flexispira, respectively) werehighly coated, suggesting that IgA coating does not strictly correlatewith anatomical location. As predicted, multiple highly coated bacteriawere indeed found in the mucus, including SFB and Helicobacter sp.flexispira, which were almost exclusively mucus-associated. However,mucus association alone was insufficient to trigger high IgA coatingsince many mucus-associated taxa were devoid of significant IgA coating(e.g., unclassified Helicobacteraceae, Mucispirillum, unclassifiedClostridiales, and Ruminococcus) (FIG. 2B).

As the initiation of a bacterial-specific IgA response requires intimateinteraction with the mucosal immune system, the taxa that wereidentified as highly coated may have broad effects on the intestinalimmune system and, therefore, disease susceptibility. Indeed, four ofthe six taxa that were identified by IgA-SEQ are well characterized asmembers of the microbiota that modulate the intestinal immune responseand thereby alter susceptibility to disease: SFB, Lactobacillus,Prevotellaceae, and Helicobacter (Hooper et al., 2012, Science 336:1268;Ivanov et al., 2009, Cell 139:485; Elinav et al., 2011, Cell 145:745).Additional investigation into the effects of the remaining IgA coatedtaxa (S24-7 and Erysipelotrichaceae) on the immune system is ongoing,although Erysipelotrichaceae species have been linked to obesity (Zhanget al., 2009, Proceedings of the National Academy of Sciences 106:2365).

The IgA coating landscape of intestinal bacteria in 20 healthy humanswas examined next. As expected, fecal bacteria from human subjectsshowed variable levels of IgA coating, as measured by flow cytometry(FIG. 10A) (van der Waaij et al., 1996, Gut 38:348; van der Waaij etal., 1994, Cytometry 16:270), and the taxonomic composition of themicrobiota varied greatly between subjects (FIG. 11). IgA coatingpatterns, as measured by ICI, also differed substantially betweenhumans—some subjects showed ICI scores greater than ten for only one ortwo bacterial species, while others showed high ICI scores for six ormore species (FIG. 12). Despite the observed variability in taxonomiccomposition and IgA coating, clustering analysis (complete linkageclustering using Euclidean distance) of ICI scores of all bacterial taxarevealed a clear pattern of IgA coating across healthy humans,demonstrating that IgA coating of specific bacterial taxa is conservedwithin the healthy population (FIG. 3A). Statistical comparisons of IgAcoated and non-coated fractions revealed six significantly IgA coatedbacterial species (P<0.05, LEfSe and Wilcoxon rank-sum) (FIG. 13A): anunclassified Bifidobacterium, Akkermansia muciniphila, Ruminococcustorques, Dorea spp., an unclassified Erysipelotricaceae, and Actinomycesspp. These taxa thus represent the most prevalent and conserved IgAcoated bacteria in healthy humans. As in mice, highly coated members ofthe microbiota may preferentially affect host health and disease throughtheir interactions with the immune system. Interestingly, while theeffects of most of the species that were identified as IgA coated arenot well characterized in the literature, Akkermansia muciniphila wasrecently found to induce mucus production and lead to weight loss inobese mice (Everard et al., 2013, Proceedings of the National Academy ofSciences of the United States of America 110:9066) and Bifidobacteriumspp. were found to induce IL-10 producing T regulatory type 1 cells inthe colon (Jeon et al., 2012, PLoS pathogens 8:e1002714).

In addition to the conserved IgA coated taxa, multiple taxa were highlycoated with IgA in one or more healthy subjects (FIG. 3A). In somecases, these taxa were present in only a single human subject (e.g.,Prevotellaceae spp.). In other cases, a taxon was broadly prevalent, butwas only coated in a single human subject (e.g., Coprococcus catus),consistent with the explanation that genetic, environmental, or uniquebacterial factors might determine whether or not these particular taxaare coated with IgA.

While it is known that interactions between the microbiota and theimmune system play a critical role in inflammatory bowel diseases (IBD),such as Crohn's Disease (CD) and Ulcerative Colitis (UC) (Strober, 2013,Trends in immunology 34:423), the identities of specificimmunomodulatory bacteria in these diseases are not known. Therefore,IgA coating in fecal samples from 27 patients with Crohn's disease and 8patients with Ulcerative Colitis was examined (FIGS. 3B and 3C, and FIG.10). As was observed in healthy controls, IBD patients exhibitedsignificant variability in their microbiota compositions (FIGS. 11B and11C). Since IBD patients exhibit chronic intestinal inflammation thatcan lead to disruption of the epithelial barrier, it was expected thatIgA coating of all taxa might be increased leading to homogenous coatingof the microbiota in IBD. However, like healthy controls, individual IBDpatients showed high coating of only a limited number of taxa. Whencompared with the pattern of IgA coating in control subjects, CD and UCpatients showed markedly increased diversity in the bacterial taxa thatwere highly coated with IgA (FIGS. 3B and 3C). Specific bacterial taxathat were always present but rarely or never coated in control patientswere highly coated in one or more IBD patients (e.g., Faecalibacteriumprausnitzii and Coprococcus spp.), which, while not wishing to be boundby any particular theory, is consistent with the explanation that IBDpatients mount immune responses to bacteria that are normally ignored.In addition, IBD patients often showed reduced coating of taxa that wereconsistently coated in control subjects, even when the relativeabundance of that taxon was not significantly altered; for example,average coating of Dorea spp. was reduced overall when comparing ICIs ofcontrol and CD patients (P<0.05, LEfSe and Wilcoxon rank-sum). Overall,IBD patients as a group showed a dramatically altered pattern of IgAcoating as compared to controls: four of the six taxa that wereconsistently coated in controls were not significantly coated in IBDpatients, and UC patients ‘exchanged’ their IgA coated unclassifiedBifidobacterium for a different Bifidobacterium spp. Furthermore, bothCD and UC patients showed high coating of members of the Streptococcusgenus, which were not coated with IgA in healthy controls (P<0.05, LEfSeand Wilcoxon rank-sum).

Multiple taxa that were uniquely present in IBD were highly coated in atleast one patient (e.g., unclassified Bulleidia, Allobaculum spp.,Lactobacillus mucosae, unclassified Pediococcus, and Weissella spp.). Inaddition, multiple species that were rarely present and never highlycoated in control subjects were highly coated in multiple IBD patients(e.g., Streptococcus luteciae, Haemophilus parainfluenzae, andLactobacillus zeae)(See Table 1). Because transmissible members of theAsc^(−/−) mouse microbiota become highly IgA coated in wild typerecipient animals, and these recipients exhibit increased sensitivity tocolitis, IBD-specific IgA coated bacteria present interesting candidatesfor bacteria that may drive inflammation in IBD.

TABLE 1 Frequency of Subjects with ICI > 10 Control CD UC Highly Coatedin UC and CD but not Control Streptococcus luteciae 0.0 14.8 12.5Coprococcus 0.0 14.8 12.5 Haemophilus parainfluenzae 0.0 11.1 37.5Faecalibacterium prausnitzii 0.0 7.4 25.0 Collinsella aerofaciens 0.07.4 12.5 Blautia obeum 0.0 7.4 12.5 Oscillospira 0.0 3.7 25.0Turicibacter 0.0 3.7 12.5 Clostridium perfringens 0.0 3.7 12.5Veillonella dispar 0.0 3.7 12.5 Highly Coated in CD but not Control orUC Blautia 0.0 14.8 0.0 Lactobacillus Other 0.0 11.1 0.0 Clostridium 0.011.1 0.0 UC Ruminococcaceae 0.0 11.1 0.0 Lactobacillus 0.0 7.4 0.0Lactobacillus reuteri 0.0 7.4 0.0 Pediococcus Other 0.0 7.4 0.0Acidaminococcus 0.0 7.4 0.0 Bacteroides Other 0.0 3.7 0.0 Bacteroides0.0 3.7 0.0 Lactobacillus mucosae 0.0 3.7 0.0 Weissella 0.0 3.7 0.0 UCClostridiales 0.0 3.7 0.0 Anaerostipes 0.0 3.7 0.0 Roseburia 0.0 3.7 0.0Roseburia faecis 0.0 3.7 0.0 Veillonella 0.0 3.7 0.0 Bulleidia Other 0.03.7 0.0 [Eubacterium] 0.0 3.7 0.0 Highly Coated in UC but not Control orCD Rikenellaceae 0.0 0.0 25.0 Lactobacillus zeae 0.0 0.0 25.0[Eubacterium] dolichum 0.0 0.0 25.0 Eggerthella lenta 0.0 0.0 12.5Ruminococcus 0.0 0.0 12.5 Allobaculum 0.0 0.0 12.5 More frequentlycoated in UC or CD than Control UC Erysipelotrichaceae 20.0 25.9 37.5Actinomyces 5.0 22.2 25.0 Streptococcus 5.0 18.5 25.0 Dialister 5.0 18.525.0 Blautia producta 5.0 18.5 12.5 Erysipelotrichaceae 10.0 14.8 50.0Bifidobacterium 20.0 14.8 37.5 [Ruminococcus] gnavus 15.0 14.8 25.0Bifidobacterium Other 25.0 11.1 50.0 Blautia Other 5.0 11.1 25.0Streptococcus Other 10.0 11.1 0.0 SMB53 10.0 11.1 0.0 Coprococcus catus5.0 7.4 0.0 Ruminococcaceae 5.0 7.4 0.0 [Eubacterium] biforme 5.0 7.40.0 Ruminococcus bromii 20.0 3.7 25.0 Bifidobacterium adolescentis 5.03.7 12.5 Lachnospiraceae_other 10.0 3.7 12.5 Bacteroides fragilis 5.00.0 50.0 Lachnospiraceae 5.0 0.0 12.5 Sutterella 5.0 0.0 12.5 Morefrequently coated in Control than UC or CD Akkermansia muciniphila 55.07.4 25.0 [Ruminococcus] torques 50.0 25.9 25.0 Dorea 40.0 18.5 0.0 Doreaformicigenerans 30.0 11.1 0.0 UC Enterobacteriaceae 30.0 22.2 12.5 UCFaecalibacterium 20.0 7.4 0.0 UC Dorea 15.0 0.0 0.0 Ruminococcuscallidus 10.0 0.0 0.0 Methanobrevibacter 10.0 3.7 0.0 Clostridiales 10.03.7 0.0 UC [Ruminococcus] 10.0 7.4 0.0 [Ruminococcus] 10.0 7.4 0.0Parabacteroides distasonis 5.0 0.0 0.0 Paraprevotella 5.0 0.0 0.0[Prevotella] 5.0 0.0 0.0 Lactococcus 5.0 0.0 0.0 Christensenellaceae 5.00.0 0.0 Coprococcus Other 5.0 0.0 0.0 Coprococcus eutactus 5.0 0.0 0.0Ruminococcus Other 5.0 0.0 0.0 Bilophila 5.0 0.0 0.0 Slackia 5.0 3.7 0.0UC Parabacteroides 5.0 3.7 0.0 Megasphaera 5.0 3.7 0.0

Many factors can potentially influence IgA coating of specific membersof the microbiota, including bacterial abundance, location, theimmunogenicity of particular bacterial epitopes, and bacterial evasionmechanisms, such as epitope masking or antigenic drift (Macpherson 2012,Immunological reviews 245:132). Furthermore, host genetics and thecomposition of the remaining members of the microbiota may also affectpatterns of IgA coating. Despite this apparent complexity, it was foundthat a limited number of taxa were highly coated with IgA in mice andhumans in both health and disease. These taxa thus preferentiallystimulate intestinal immune responses, possibly because they penetratethe mucus barrier and interact directly with cells of the mucosal immunesystem. Indeed, many of the highly coated taxa identified in mice areknown to have broad effects on the immune system and, therefore, ondisease susceptibility. Importantly, the immunomodulatory effects ofalmost all of the highly coated bacteria that were identified in humansremain unexplored. Integrating 16S-based microbiota profiling withtaxa-specific information regarding the host immune response may enhancepredictions of disease susceptibility and enable personalized approachesto disease prevention and treatment.

Experiments were conducted to evaluate and compare the IgA coating offecal bacteria in healthy and obese adolescents. Depicted in the mainheatmap are IgA coating index (ICI) scores for bacterial species from 4healthy and 15 obese adolescents (FIG. 14). Each column represents anindividual human subject. Bacterial taxa are clustered (complete linkageclustering using Euclidean distance) based ICI scores. Bacterial taxafrom obese patients with significantly higher relative abundance in theIgA+ fraction as compared to the IgA− fraction by LEfSe are consideredto be highly coated with IgA and are labeled in red (###) (FIG. 14).

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A method of identifying a type of bacteria in themicrobiota of a subject that contributes to the development orprogression of an inflammatory disease or disorder in the subject, themethod comprising the steps of: a. isolating a secretory antibody-boundbacteria from the subject's biological sample, b. amplifying bacterialnucleic acid from the secretory antibody-bound bacteria so isolated, c.determining the sequences of the amplicons so amplified, d. identifyingthe type of antibody-bound bacteria present in the subject's biologicalsample by identifying nucleic acid sequences that are indicative ofparticular types of bacteria.
 2. The method of claim 1, wherein themicrobiota of the subject is on or near a mucosal surface of the subjectselected from the group consisting of the gastrointestinal tract, therespiratory tract, genitourinary tract and mammary gland.
 3. The methodof claim 1, wherein the biological sample is at least one of a fecalsample, a mucus sample, a sputum sample, and a breast milk sample. 4.The method of claim 1, wherein the bacterial nucleic acid is 16S rRNA.5. The method of claim 1, wherein the secretory antibody is at least oneselected from the group consisting of IgA1, IgA2, and IgM.
 6. The methodof claim 1, wherein the type of bacteria identified in the biologicalsample is at least one selected from the group consisting of SegmentedFilamentous Bacteria (SFB) and Helicobacter flexispira.
 7. The method ofclaim 1, wherein the type of bacteria identified in the biologicalsample is at least one selected from the group consisting ofLactobacillus, Helicobacter, S24-7, Erysipelotrichaceae, Prevotellaceae,Eubacterium, Eubacterium biforme, Eubacterium dolichum, Ruminococcusgnavus, Acidaminococcus, Actinomyces, Allobaculum, Anaerostipes,Bacteroides, Bacteroides fragilis, Bacteroides Other, Bifidobacterium,Bifidobacterium adolescentis, Bifidobacterium Other, Blautia, Blautiaobeum, Blautia Other, Blautia producta, Bulleidia Other, Clostridium,Clostridium perfringens, Collinsella aerofaciens, Coprococcus,Coprococcus catus, Dialister, Eggerthella lenta, Erysipelotrichaceae,Faecalibacterium prausnitzii, Haemophilus parainfluenzae,Lachnospiraceae, Lachnospiraceae other, Lactobacillus, Lactobacillusmucosae, Lactobacillus Other, Lactobacillus reuteri, Lactobacillus zeae,Oscillospira, Pediococcus Other, Rikenellaceae, Roseburia, Roseburiafaecis, Ruminococcaceae, Ruminococcus, Ruminococcus bromii, SMB53,Streptococcus, Streptococcus luteciae, Streptococcus Other, Sutterella,Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UCRuminococcaceae, Veillonella, Veillonella dispar, and Weissella.
 8. Themethod of claim 7, wherein the bacteria from the bacterial familyPrevotellaceae is a bacteria from the bacterial genera of Paraprevotellaor Prevotella.
 9. The method of claim 1, wherein the inflammatorydisease or disorder is at least one inflammatory disease or disorderselected from the group consisting of inflammatory bowel disease, celiacdisease, colitis, intestinal hyperplasia, metabolic syndrome, obesity,rheumatoid arthritis, liver disease, hepatic steatosis, fatty liverdisease, non-alcoholic fatty liver disease (NAFLD), and non-alcoholicsteatohepatitis (NASH).
 10. The method of claim 1, wherein the subjectis human.
 11. A method of diagnosing an inflammatory disease or disorderin a subject in need thereof by identifying a type of bacteria in themicrobiota of the subject that contributes to the development orprogression of an inflammatory disease or disorder, the methodcomprising the steps of: a. isolating secretory an antibody-boundbacteria from the subject's biological sample, b. amplifying bacterialnucleic acid from the secretory antibody-bound bacteria so isolated, c.determining the sequences of the amplicons so amplified, d. identifyingthe type of antibody-bound bacteria present in the subject's biologicalsample by identifying nucleic acid sequences that are indicative ofparticular types of bacteria. wherein when the type of antibody-boundbacteria present in the subject's biological sample is a type ofbacteria that contributes to the development or progression of aninflammatory disease or disorder, the subject is diagnosed with theinflammatory disease or disorder.
 12. The method of claim 11, whereinthe microbiota of the subject is on or near a mucosal surface of thesubject selected from the group consisting of the gastrointestinaltract, the respiratory tract, genitourinary tract and mammary gland. 13.The method of claim 11, wherein the biological sample is at least one ofa fecal sample, a mucus sample, a sputum sample, and a breast milksample.
 14. The method of claim 11, wherein the bacterial nucleic acidis 16S rRNA.
 15. The method of claim 11, wherein the secretory antibodyis at least one selected from the group consisting of IgA1, IgA2, andIgM.
 16. The method of claim 11, wherein the type of bacteria identifiedin the biological sample is at least one selected from the groupconsisting of Segmented Filamentous Bacteria (SFB) and Helicobacterflexispira.
 17. The method of claim 11, wherein the type of bacteriaidentified in the biological sample is at least one selected from thegroup consisting of Lactobacillus, Helicobacter, S24-7,Erysipelotrichaceae, Prevotellaceae, Eubacterium, Eubacterium biforme,Eubacterium dolichum, Ruminococcus gnavus, Acidaminococcus, Actinomyces,Allobaculum, Anaerostipes, Bacteroides, Bacteroides fragilis,Bacteroides Other, Bifidobacterium, Bifidobacterium adolescentis,Bifidobacterium Other, Blautia, Blautia obeum, Blautia Other, Blautiaproducta, Bulleidia Other, Clostridium, Clostridium perfringens,Collinsella aerofaciens, Coprococcus, Coprococcus catus, Dialister,Eggerthella lenta, Erysipelotrichaceae, Faecalibacterium prausnitzii,Haemophilus parainfluenzae, Lachnospiraceae, Lachnospiraceae other,Lactobacillus, Lactobacillus mucosae, Lactobacillus Other, Lactobacillusreuteri, Lactobacillus zeae, Oscillospira, Pediococcus Other,Rikenellaceae, Roseburia, Roseburia faecis, Ruminococcaceae,Ruminococcus, Ruminococcus bromii, SMB53, Streptococcus, Streptococcusluteciae, Streptococcus Other, Sutterella, Turicibacter, UCClostridiales, UC Erysipelotrichaceae, UC Ruminococcaceae, Veillonella,Veillonella dispar, and Weissella.
 18. The method of claim 17, whereinthe bacteria from the bacterial family Prevotellaceae is a bacteria fromthe bacterial genera of Paraprevotella or Prevotella.
 19. The method ofclaim 11, wherein the inflammatory disease or disorder is at least oneinflammatory disease or disorder selected from the group consisting ofinflammatory bowel disease, celiac disease, colitis, intestinalhyperplasia, metabolic syndrome, obesity, rheumatoid arthritis, liverdisease, hepatic steatosis, fatty liver disease, non-alcoholic fattyliver disease (NAFLD), and non-alcoholic steatohepatitis (NASH).
 20. Themethod of claim 11, wherein the subject is human.
 21. A method oftreating an inflammatory disease or disorder associated with a secretoryantibody-bound bacteria in the microbiota of a subject in need thereof,the method comprising administering to the subject at least one therapyto diminish the number of at least one type of bacteria that isover-represented in the microbiota of the subject.
 22. The method ofclaim 21, wherein the at least one therapy is at least one selected fromthe group consisting of at least one vaccine, at least one antibiotic,and at least one passive immunotherapy.
 23. The method of claim 21,wherein the microbiota of the subject is on or near a mucosal surface ofthe subject selected from the group consisting of the gastrointestinaltract, the respiratory tract, genitourinary tract and mammary gland. 24.The method of claim 21, wherein the biological sample is at least one ofa fecal sample, a mucus sample, a sputum sample, and a breast milksample.
 25. The method of claim 21, wherein the secretory antibody is atleast one selected from the group consisting of IgA1, IgA2, and IgM. 26.The method of claim 21, wherein the type of bacteria is at least oneselected from the group consisting of Segmented Filamentous Bacteria(SFB) and Helicobacter flexispira.
 27. The method of claim 21, whereinthe type of bacteria is at least one selected from the group consistingof Lactobacillus, Helicobacter, S24-7, Erysipelotrichaceae,Prevotellaceae, Eubacterium, Eubacterium biforme, Eubacterium dolichum,Ruminococcus gnavus, Acidaminococcus, Actinomyces, Allobaculum,Anaerostipes, Bacteroides, Bacteroides fragilis, Bacteroides Other,Bifidobacterium, Bifidobacterium adolescentis, Bifidobacterium Other,Blautia, Blautia obeum, Blautia Other, Blautia producta, BulleidiaOther, Clostridium, Clostridium perfringens, Collinsella aerofaciens,Coprococcus, Coprococcus catus, Dialister, Eggerthella lenta,Erysipelotrichaceae, Faecalibacterium prausnitzii, Haemophilusparainfluenzae, Lachnospiraceae, Lachnospiraceae other, Lactobacillus,Lactobacillus mucosae, Lactobacillus Other, Lactobacillus reuteri,Lactobacillus zeae, Oscillospira, Pediococcus Other, Rikenellaceae,Roseburia, Roseburia faecis, Ruminococcaceae, Ruminococcus, Ruminococcusbromii, SMB53, Streptococcus, Streptococcus luteciae, StreptococcusOther, Sutterella, Turicibacter, UC Clostridiales, UCErysipelotrichaceae, UC Ruminococcaceae, Veillonella, Veillonelladispar, and Weissella.
 28. The method of claim 26, wherein the bacteriafrom the bacterial family Prevotellaceae is a bacteria from thebacterial genera of Paraprevotella or Prevotella.
 29. The method ofclaim 21, wherein the inflammatory disease or disorder is at least oneinflammatory disease or disorder selected from the group consisting ofinflammatory bowel disease, celiac disease, colitis, intestinalhyperplasia, metabolic syndrome, obesity, rheumatoid arthritis, liverdisease, hepatic steatosis, fatty liver disease, non-alcoholic fattyliver disease (NAFLD), and non-alcoholic steatohepatitis (NASH).
 30. Themethod of claim 21, wherein the subject is human.
 31. The method ofclaim 21, wherein the therapy induces an immune response directedagainst at least one type of secretory antibody-bound bacteria presentin the microbiota of the subject
 32. The method of claim 22, wherein theat least one antibiotic is at least one selected from the groupconsisting of lipopeptide, fluoroquinolone, ketolide, cephalosporin,amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,streptomycin, tobramycin, cefacetrile, cefadroxil, cefalexin,cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin,cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine,ceftezole, cefaclor, cefamandole, cefmetazole, cefonicid, cefotetan,cefoxitin, cefprozil, cefuroxime, cefuzonam, cefcapene, cefdaloxime,cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime,cefotaxime, cefpimizole, cefpodoxime, cefteram, ceftibuten, ceftiofur,ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime,cefclidine, cefepime cefluprenam, cefoselis, cefozopran, cefpirome,cefquinome, cefaclomezine, cefaloram, cefaparole, cefcanel, cefedrolor,cefempidone, cefetrizole, cefivitril, cefmatilen, cefmepidium,cefovecin, cefoxazole, cefrotil, cefsumide, ceftaroline, ceftioxide,cefuracetime, imipenem, primaxin, doripenem, meropenem, ertapenem,flumequine, nalidixic acid, oxolinic acid, piromidic acid pipemidicacid, rosoxacin, ciprofloxacin, enoxacin, lomefloxacin, nadifloxacin,norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin,gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin,sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gemifloxacin,sitafloxacin, trovafloxacin, prulifloxacin, azithromycin, erythromycin,clarithromycin, dirithromycin, roxithromycin, telithromycin,amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin,dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin,penicillin g, penicillin v, piperacillin, pivampicillin, pivmecillinam,ticarcillin, sulfamethizole, sulfamethoxazole, sulfisoxazole,trimethoprim-sulfamethoxazole, demeclocycline, doxycycline, minocycline,oxytetracycline, tetracycline, linezolid, clindamycin, metronidazole,vancomycin, vancocin, mycobutin, rifampin, nitrofurantoin,chloramphenicol, or derivatives thereof.
 33. The method of claim 21,wherein the method further comprises administering to the subject atleast probiotic to increase the number of at least one type of bacteriaunder-represented in the microbiota of the subject.